Methods for improved production of rebaudioside d and rebaudioside m

ABSTRACT

Methods for recombinant production of steviol glycoside and compositions containing steviol glycosides are provided by this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of the U.S. application Ser. No.15/908,214, filed on Feb. 28, 2018, and issued as the U.S. Pat. No.10,612,066 on Apr. 7, 2020, which is a divisional of the U.S.application Ser. No. 14/761,629, filed on Jul. 17, 2015, and issued asthe U.S. Pat. No. 9,957,540 on May 1, 2018, which is the U.S. NationalStage Application of International Application No. PCT/EP2014/052363,filed on Feb. 6, 2014, and claims the benefit of the U.S. ProvisionalApplication No. 61/761,490, filed on Feb. 6, 2013 and the U.S.Provisional Application No. 61/886,442, filed on Oct. 3, 2013, thedisclosures of each of which are explicitly incorporated by referenceherein in their entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The invention disclosed herein relates generally to the field ofrecombinant production of steviol glycosides. Particularly, theinvention provides methods for recombinant production of steviolglycoside and compositions containing steviol glycosides.

Description of Related Art

Sweeteners are well known as ingredients used most commonly in the food,beverage, or confectionary industries. The sweetener can either beincorporated into a final food product during production or forstand-alone use, when appropriately diluted or as a tabletop sweetener.Sweeteners include natural sweeteners such as sucrose, high fructosecorn syrup, molasses, maple syrup, and honey and artificial sweetenerssuch as aspartame, saccharine and sucralose. Stevia extract is a naturalsweetener that can be isolated and extracted from a perennial shrub,Stevia rebaudiana. Stevia is commonly grown in South America and Asiafor commercial production of stevia extract. Stevia extract, purified tovarious degrees, is used commercially as a high intensity sweetener infoods and in blends or alone as a tabletop sweetener.

Extracts of the Stevia plant contain Rebaudiosides and other steviolglycosides that contribute to the sweet flavor, although the amount ofeach glycoside often varies among different production batches. Existingcommercial products are predominantly Rebaudioside A with lesser amountsof other glycosides such as Rebaudioside C, D, and F. Stevia extractscan also contain contaminants such as plant-derived compounds thatcontribute to off-flavors or have other undesirable effects. Thesecontaminants can be more or less problematic depending on the foodsystem or application of choice. Potential contaminants includepigments, lipids, proteins, phenolics, saccharides, spathulenol andother sesquiterpenes, labdane diterpenes, monoterpenes, decanoic acid,8,11,14-eicosatrienoic acid, 2-methyloctadecane, pentacosane,octacosane, tetracosane, octadecanol, stigmasterol, beta-sitosterol,alpha- and beta-amyrin, lupeol, beta-amryin acetate, pentacyclictriterpenes, centauredin, quercitin, epi-alpha-cadinol, carophyllenesand derivatives, beta-pinene, beta-sitosterol, and gibberellin.

As recovery and purification of steviol glycosides from the Stevia planthave proven to be labor intensive and inefficient, there remains a needfor a recombinant production system that can produce high yields ofdesired steviol glycosides such as Rebaudioside D and Rebaudioside Mwith less plant-based contaminants, including but not limited tostevioside. Steviol glycoside-producing Saccharomyces cerevisiae strainsas well as bio-conversion and biosynthesis in vitro are described in PCTApplication Nos. PCT/US2012/050021 and PCT/US2011/038967, which areincorporated herein by reference in their entirety.

In nature, the Stevia uridine diphosphate dependent glycosyltransferase76G1 (UGT76G1) catalyzes several glycosylation reactions on the steviolbackbone, which leads to the production of steviol glycosides. Recently,it has been shown that UGT76G1 can convert 1,2-stevioside toRebaudioside A and 1,2-bioside to Rebaudioside B (see Richman et al.,2005, The Plant Journal 41:56-67). Thus, there is a need in the art toidentify reactions directed towards producing glycosylated Rebaudiosidesby UGT76G1 or other UGT enzymes. Particularly, there is a need toexplore or identify other reactions catalysed by UGT76G1 as well a needto increase UGT76G1's catalytic capability in order to produce higheryields of steviol glycosides such as Rebaudioside D and Rebaudioside M.

SUMMARY OF THE INVENTION

It is against the above background that the present invention providescertain advantages and advancements over the prior art.

In particular, the invention is directed to biosynthesis of RebaudiosideD and Rebaudioside M and Rebaudioside D and Rebaudioside M preparationsfrom genetically modified cells.

In particular embodiments, the invention is directed to Rebaudioside Dand Rebaudioside M preparations from genetically modified cells havingsignificantly improved biosynthesis rates and yields.

This disclosure relates to the production of steviol glycosides. Inparticular, this disclosure relates to the production of steviolglycosides including Rebaudioside M:

-   (2S,3R,4S,5R,6R)-5-hydroxy-6-(hydroxymethyl)-3,4-bis({[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy})oxan-2-yl(1R,5R,9S,13R)-13-{[(2S,3R,4S,5R,6R)-5-hydroxy-6-(hydroxymethyl)-3,4-bis({[(2S,3R,4S,5S,6R)-3,4,5-tri    hydroxy-6-(hydroxymethyl)oxan-2-yl]oxy})oxan-2-yl]oxy}-5,9-dimethyl-14-methylidenetetracyclo[11.2.1.0^(1,10)0.0^(4,9)]    hexadecane-5-carboxylate-   and Rebaudioside D:-   4,5-dihydroxy-6-(hydroxymethyl)-3-{[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}oxan-2-yl    13-{[5-hydroxy-6-(hydroxymethyl)-3,4-bis({[3,4,5-trihydroxy-6-(hydroxymethyl)    oxan-2-yl]oxy})oxan-2-yl]oxy}-5,9-dimethyl-14-methylidene    tetracyclo[11.2.1.^(01,10)0.0^(4,9)] hexadecane-5-carboxylate-   by means not limited to in recombinant hosts such as recombinant    microorganisms, through bioconversion, and in vitro.

Thus, in one aspect, the disclosure provides a recombinant host, forexample, a microorganism, comprising one or more biosynthetic genes,wherein the expression of one or more biosynthetic genes results inproduction of steviol glycosides including Rebaudioside M andRebaudioside D.

In particular, expression of one or more uridine 5′-diphospho (UDP)glycosyl transferases described herein, such as EUGT11, UGT74G1,UGT76G1, UGT85C2, and UGT91D2, facilitate production and accumulation ofRebaudioside M or Rebaudioside D in recombinant hosts or certain invitro systems.

Although this invention disclosed herein is not limited to specificadvantages or functionality, the invention provides a compositioncomprising from about 1% to about 99% w/w of Rebaudioside M, wherein thecomposition has a reduced level of Stevia-derived contaminants relativeto a stevia extract, wherein at least one of said contaminants is aplant-derived compound. In certain instances, said plant-derivedcontaminating compound can, inter alia, contribute to off-flavors.

In some aspects, the composition comprising from about 1% to about 99%w/w of Rebaudioside M has less than 0.1% of Stevia-derived contaminantsrelative to a stevia extract, wherein at least one of said contaminantsis a plant-derived compound. In certain instances, said plant-derivedcontaminating compound can, inter alia, contribute to off-flavors.

The invention further provides a food product comprising the compositionas described above.

In some aspects, the food product is a beverage or a beverageconcentrate.

The invention further provides a recombinant host cell that expresses:

(a) a recombinant gene encoding a GGPPS;

(b) a recombinant gene encoding an ent-copalyl diphosphate synthase(CDPS) polypeptide;

(c) a recombinant gene encoding a kaurene oxidase (KO) polypeptide;

(d) a recombinant gene encoding a kaurene synthase (KS) polypeptide;

(e) a recombinant gene encoding a steviol synthase (KAH) polypeptide;

(f) a recombinant gene encoding a cytochrome P450 reductase (CPR)polypeptide;

(g) a recombinant gene encoding a UGT85C2 polypeptide;

(h) a recombinant gene encoding a UGT74G1 polypeptide;

(i) a recombinant gene encoding a UGT76G1 polypeptide;

(j) a recombinant gene encoding a UGT91d2 polypeptide; and

(k) a recombinant gene encoding a EUGT11 polypeptide;

wherein at least one of said genes is a recombinant gene and wherein thecell produces Rebaudioside D, Rebaudioside M, Rebaudioside Q, and/orRebaudioside I.

The invention further provides a recombinant host cell comprisingexogenous nucleic acids comprising:

(a) a recombinant gene encoding a GGPPS;

(b) a recombinant gene encoding an ent-copalyl diphosphate synthase(CDPS) polypeptide;

(c) a recombinant gene encoding a kaurene oxidase (KO) polypeptide;

(d) a recombinant gene encoding a kaurene synthase (KS) polypeptide;

(e) a recombinant gene encoding a steviol synthase (KAH) polypeptide;

(f) a recombinant gene encoding a cytochrome P450 reductase (CPR)polypeptide;

(g) a recombinant gene encoding a UGT85C2 polypeptide;

(h) a recombinant gene encoding a UGT74G1 polypeptide;

(i) a recombinant gene encoding a UGT76G1 polypeptide;

(j) a recombinant gene encoding a UGT91d2 polypeptide; and

(k) a recombinant gene encoding a EUGT11 polypeptide;

wherein the cell produces Rebaudioside D, Rebaudioside M, RebaudiosideQ, and/or Rebaudioside I.

The invention further provides a recombinant host cell that expresses aGGPPS, an ent-copalyl diphosphate synthase (CDPS) polypeptide, a kaureneoxidase (KO) polypeptide, a kaurene synthase (KS) polypeptide; a steviolsynthase (KAH) polypeptide, a cytochrome P450 reductase (CPR)polypeptide, a UGT74G1 polypeptide, a UGT76G1 polypeptide, a UGT91d2polypeptide, and a EUGT11 polypeptide, wherein at least one of saidpolypeptides is encoded by an exogenous or heterologous gene having beenintroduced into said cell;

wherein the cell produces a di-glycosylated steviol glycoside(13-hydroxy kaur-16-en-18-oic acid,[2-O-β-D-glucopyranosyl-β-D-glucopyranosyl] ester) or a tri-glycosylatedsteviol glycoside (13-hydroxy kaur-16-en-18-oic acid;[2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester).

In some embodiments, targeted production of individual Rebaudiosides canbe accomplished by controlling the relative levels of UDP-glycosyltransferase activities (see FIG. 1).

In some aspects, targeted production of individual Rebaudiosides can beaccomplished by differential copy numbers of the UGT-encoding genes (seeFIG. 1) in the recombinant cell, differential promoter strengths, and/orby utilizing mutants with increased specificity/activity towards theproduct of interest. For example, low levels of Rebaudioside D, E, and Mwill be formed if EUGT11 is expressed at low levels in comparison to theother UGTs, which would favor Rebaudioside A formation. High levels ofEUGT11 expression result in production of more 19-O 1,2 diglucoside thatcan serve as substrate for UGT76G1 to form Rebaudioside M. In certainadvantageous embodiments, additional copies or mutant versions ofUGT76G1 in recombinant cells of the invention can improve the rate ofRebaudioside M formation from Rebaudioside D.

In some embodiments, UGT76G1 catalyzes glycosylation of steviol andsteviol glycosides at the 19-O position. Thus, in some embodiments, oneor more of RebM, RebQ, Rebl, di-glycosylated steviol glycoside(13-hydroxy kaur-16-en-18-oic acid,[2-O-β-D-glucopyranosyl-β-D-glucopyranosyl] ester), or tri-glycosylatedsteviol glycoside ((13-hydroxy kaur-16-en-18-oic acid;[2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester)are produced in a recombinant host expressing a recombinant geneencoding a UGT76G1 polypeptide, through bioconversion, or throughcatalysis by UGT76G1 in vitro. In some embodiments, UGT76G1 catalyzesthe glycosylation of steviol and steviol glycosides at the 13-O positionand preferentially glycosylates steviol glycoside substrates that are1,2-di-glycosylated at the 13-O position or mono-glycosylated at the13-O position. In some embodiments, UGT76G1 does not show a preferencefor the glycosylation state of the 19-O position.

In some aspects, the GGPPS comprises Synechococcus sp. GGPPS set forthin SEQ ID NO:24.

In some aspects, the CDP polypeptide comprises a Z. mays CDPSpolypeptide set forth in SEQ ID NO:13, wherein the polypeptide islacking a chloroplast transit peptide.

In some aspects, the KO polypeptide comprises a KO polypeptide having70% or greater identity to the amino acid sequence of the S. rebaudianaKO polypeptide set forth in SEQ ID NO:25.

In some aspects, the KS polypeptide comprises a KS polypeptide having40% or greater identity to the amino acid sequence of the A. thaliana KSpolypeptide set forth in SEQ ID NO:21.

In some aspects, the KAH polypeptide comprises a KAH polypeptide having60% or greater identity to the S. rebaudiana KAH amino acid sequence setforth in SEQ ID NO:11.

In some aspects, the CPR polypeptide comprises a CPR polypeptide having65% or greater identity to a S. rebaudiana CPR amino acid sequence setforth in SEQ ID NO:4, an A. thaliana CPR polypeptide of the amino acidsequence set forth in SEQ ID NO:9 or a combination thereof.

In some aspects, the UGT85C2 polypeptide comprises a UGT85C2 polypeptidehaving 55% or greater identity to the amino acid sequence set forth inSEQ ID NO:26.

In some aspects, the UGT74G1 polypeptide comprises a UGT74G1 polypeptidehaving 55% or greater identity to the amino acid sequence set forth inSEQ ID NO:19.

In some aspects, the UGT76G1 polypeptide comprises a UGT76G1 polypeptidehaving 50% or greater identity to the amino acid sequence set forth inSEQ ID NO:2.

In some aspects, the UGT91d2 polypeptide comprises a UGT91d2 polypeptidehaving 90% or greater identity to the amino acid sequence set forth inSEQ ID NO:26 or a functional homolog thereof, a UGT91d2e polypeptidehaving a substitution at residues 211 and 286 of SEQ ID NO:15 or acombination thereof.

In some aspects, the EUGT11 polypeptide comprises a EUGT11 polypeptidehaving 65% or greater identity to the Os03g0702000 amino acid sequenceset forth in SEQ ID NO:16.

In some aspects, the UGT76G1 polypeptide comprises one or more of theUGT76G1 polypeptide variants comprising: T55K, T55E, S56A, Y128S, Y128E,H155L, H155R, Q198R, S285R, S285T, S253W, S253G, T284R, T284G, S285G,K337E, K337P and L379V of SEQ ID NO:2.

In some aspects, the UGT76G1 polypeptide comprises one or more of theUGT76G1 polypeptide variants comprising: Q23G, Q23H, I26F, I26W, T146A,T146G, T146P, H155R, L257P, L257W, L257T, L257G, L257A, L257R, L257E,S283G and S283N of SEQ ID NO:2.

In some aspects, the recombinant host cell is a yeast cell, a plantcell, a mammalian cell, an insect cell, a fungal cell or a bacterialcell.

In some aspects, the yeast cell is a cell from Saccharomyces cerevisiae,Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbyagossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis,Hansenula polymorpha, Candida boidinii, Arxula adeninivorans,Xanthophyllomyces dendrorhous or Candida albicans species.

In some aspects, the yeast cell is a Saccharomycete.

In some aspects, the yeast cell is a cell from Saccharomyces cerevisiaespecies.

The invention further provides the cell as disclosed herein thatproduces Rebaudioside D.

The invention further provides the cell as disclosed herein thatproduces Rebaudioside M, Rebaudioside Q or Rebaudioside I.

The invention further provides the cell as disclosed herein thatproduces the di-glycosylated steviol glycoside (13-hydroxykaur-16-en-18-oic acid, [2-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester) or the tri-glycosylated steviol glycoside (13-hydroxykaur-16-en-18-oic acid;[2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester).

In some aspects, the Rebaudioside D is produced in the cell as disclosedherein at a concentration of between about 1,000 mg/L and about 2,900mg/L.

In some aspects, the Rebaudioside D and Rebaudioside M are produced inthe cell as disclosed herein at a ratio of between about 1:1 to about1.7:1.

In some aspects, the Rebaudioside M is produced in the cell as disclosedherein at a concentration of between about 600 mg/L and about 2,800mg/L.

In some aspects, the Rebaudioside M and Rebaudioside D are produced inthe cell as disclosed herein at a ratio of between about 0.6:1 to about1.1:1.

The invention further provides a method of producing Rebaudioside D,Rebaudioside M, Rebaudioside Q, Rebaudioside I, di-glycosylated steviolglycoside (13-hydroxy kaur-16-en-18-oic acid,[2-O-β-D-glucopyranosyl-β-D-glucopyranosyl] ester) or tri-glycosylatedsteviol glycoside (13-hydroxy kaur-16-en-18-oic acid;[2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester), comprising:

(a) culturing a recombinant cell in a culture medium, under conditionswherein genes encoding a GGPPS; an ent-copalyl diphosphate synthase(CDPS) polypeptide; a kaurene oxidase (KO) polypeptide; a kaurenesynthase (KS) polypeptide; a steviol synthase (KAH) polypeptide; acytochrome P450 reductase (CPR) polypeptide; a UGT85C2 polypeptide; aUGT74G1 polypeptide; a UGT76G1 polypeptide; a UGT91d2 polypeptide; and aEUGT11 polypeptide are expressed, comprising inducing expression of saidgenes or constitutively expressing said genes; and

(b) synthesizing Rebaudioside D, Rebaudioside M, Rebaudioside Q,Rebaudioside I, di-glycosylated steviol glycoside (13-hydroxykaur-16-en-18-oic acid, [2-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester) or tri-glycosylated steviol glycoside (13-hydroxykaur-16-en-18-oic acid;[2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester) in the cell; and optionally

(c) isolating Rebaudioside D, Rebaudioside M, Rebaudioside Q,Rebaudioside I, di-glycosylated steviol glycoside (13-hydroxykaur-16-en-18-oic acid, [2-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester) or tri-glycosylated steviol glycoside (13-hydroxykaur-16-en-18-oic acid;[2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester).

In some aspects, Rebaudioside D is produced by a cell as disclosedherein.

In some aspects, Rebaudioside M, Rebaudioside Q or Rebaudioside I isproduced by a cell as disclosed herein.

In some aspects, di-glycosylated steviol glycoside (13-hydroxykaur-16-en-18-oic acid, [2-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester) or the tri-glycosylated steviol glycoside (13-hydroxykaur-16-en-18-oic acid;[2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester) is produced by a cell as disclosed herein.

In some aspects, Rebaudioside D is produced at a concentration ofbetween about 1,000 mg/L and about 2,900 mg/L.

In some aspects, Rebaudioside D and Rebaudioside M are produced at aratio of between about 1:1 to about 1.7:1.

In some aspects, Rebaudioside M is produced at a concentration ofbetween about 600 mg/L and about 2,800 mg/L.

In some aspects, Rebaudioside M and Rebaudioside D are produced at aratio of between about 0.6:1 to about 1.1:1.

In some aspects, a cell for practicing the methods disclosed herein is ayeast cell, a plant cell, a mammalian cell, an insect cell, a fungalcell or a bacterial cell.

In some aspects, the yeast cell is a cell from Saccharomyces cerevisiae,Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbyagossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis,Hansenula polymorpha, Candida boidinii, Arxula adeninivorans,Xanthophyllomyces dendrorhous or Candida albicans species.

In some aspects, the yeast cell is a Saccharomycete.

In some aspects, the yeast cell is a cell from Saccharomyces cerevisiaespecies.

The invention further provides methods for producing Rebaudioside D,Rebaudioside M, Rebaudioside Q, Rebaudioside I, di-glycosylated steviolglycoside (13-hydroxy kaur-16-en-18-oic acid,[2-O-β-D-glucopyranosyl-β-D-glucopyranosyl] ester) or tri-glycosylatedsteviol glycoside (13-hydroxy kaur-16-en-18-oic acid;[2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester) through in vitro bioconversion of plant-derived or syntheticsteviol or steviol glycosides using one or more UGT polypeptides.

In some aspects, said methods for producing Rebaudioside D orRebaudioside M comprise using at least one UGT polypeptide that is:

a UGT85C2 polypeptide comprising a UGT85C2 polypeptide having 55% orgreater identity to the amino acid sequence set forth in SEQ ID NO:26;

a UGT74G1 polypeptide comprising a UGT74G1 polypeptide having 55% orgreater identity to the amino acid sequence set forth in SEQ ID NO:19;

a UGT76G1 polypeptide comprising a UGT76G1 polypeptide having 50% orgreater identity to the amino acid sequence set forth in SEQ ID NO:2;

a UGT91d2 polypeptide comprising a UGT91d2 polypeptide having 90% orgreater identity to the amino acid sequence set forth in SEQ ID NO:26 ora functional homolog thereof,

a UGT91d2e polypeptide having a substitution at residues 211 and 286 ofSEQ ID NO:15 or a combination thereof; or

a EUGT11 polypeptide comprising a EUGT11 polypeptide having 65% orgreater identity to the Os03g0702000 amino acid sequence set forth inSEQ ID NO:16.

In some aspects, the steviol glycoside used for production ofRebaudioside D comprises stevioside, RebA, RebB, RebE or mixturesthereof.

In some aspects, the steviol glycoside used for production ofRebaudioside M comprises stevioside, RebA, RebB, RebE, RebD or mixturesthereof.

In some aspects, methods for producing Rebaudioside Q comprise using atleast one UGT polypeptide that is:

a UGT85C2 polypeptide comprising a UGT85C2 polypeptide having 55% orgreater identity to the amino acid sequence set forth in SEQ ID NO:26; aUGT74G1 polypeptide comprising a UGT74G1 polypeptide having 55% orgreater identity to the amino acid sequence set forth in SEQ ID NO:19;or a UGT76G1 polypeptide comprising a UGT76G1 polypeptide having 50% orgreater identity to the amino acid sequence set forth in SEQ ID NO:2.

In some aspects, the steviol glycoside used for producing Rebaudioside Qcomprises rubusoside, RebG or mixtures thereof.

In some aspects, methods for producing Rebaudioside I comprise using atleast one UGT polypeptide that is:

a UGT85C2 polypeptide comprising a UGT85C2 polypeptide having 55% orgreater identity to the amino acid sequence set forth in SEQ ID NO:26; aUGT74G1 polypeptide comprising a UGT74G1 polypeptide having 55% orgreater identity to the amino acid sequence set forth in SEQ ID NO:19; aUGT76G1 polypeptide comprising a UGT76G1 polypeptide having 50% orgreater identity to the amino acid sequence set forth in SEQ ID NO:2; aUGT91d2 polypeptide comprising a UGT91d2 polypeptide having 90% orgreater identity to the amino acid sequence set forth in SEQ ID NO:26 ora functional homolog thereof, a UGT91d2e polypeptide having asubstitution at residues 211 and 286 of SEQ ID NO:15; or a combinationthereof.

In some aspects, the steviol glycoside used for producing Rebaudioside Icomprises 1,2-stevioside, RebA, or mixtures thereof.

In some aspects, methods for producing di-glycosylated steviol glycoside(13-hydroxy kaur-16-en-18-oic acid,[2-O-β-D-glucopyranosyl-β-D-glucopyranosyl] ester) comprise using atleast one or UGT polypeptide that is:

a UGT74G1 polypeptide comprising a UGT74G1 polypeptide having 55% orgreater identity to the amino acid sequence set forth in SEQ ID NO:19;or a EUGT11 polypeptide comprising a EUGT11 polypeptide having 65% orgreater identity to the Os03g0702000 amino acid sequence set forth inSEQ ID NO:16.

In some aspects, the steviol glycoside used for producingdi-glycosylated steviol glycoside (13-hydroxy kaur-16-en-18-oic acid,[2-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester) comprisessteviol-19-O-glucoside.

In some aspects, methods for producing tri-glycosylated steviolglycoside (13-hydroxy kaur-16-en-18-oic acid;[2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester) comprise using at least one UGT polypeptide that is:

a UGT76G1 polypeptide comprising a UGT76G1 polypeptide having 50% orgreater identity to the amino acid sequence set forth in SEQ ID NO:2; aUGT74G1 polypeptide comprising a UGT74G1 polypeptide having 55% orgreater identity to the amino acid sequence set forth in SEQ ID NO:19;or a EUGT11 polypeptide comprising a EUGT11 polypeptide having 65% orgreater identity to the Os03g0702000 amino acid sequence set forth inSEQ ID NO:16.

In some aspects, the steviol glycoside used for producingtri-glycosylated steviol glycoside (13-hydroxy kaur-16-en-18-oic acid;[2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester) comprises di-glycosylated steviol glycoside (13-hydroxykaur-16-en-18-oic acid, [2-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester), steviol-19-O-glucoside, or mixtures thereof.

In some aspects, bioconversion methods as disclosed herein compriseenzymatic bioconversion or whole cell bioconversion.

In some aspects, a cell for practicing the methods disclosed herein is ayeast cell, a plant cell, a mammalian cell, an insect cell, a fungalcell or a bacterial cell.

In some aspects, the yeast cell is a cell from Saccharomyces cerevisiae,Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbyagossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis,Hansenula polymorpha, Candida boidinii, Arxula adeninivorans,Xanthophyllomyces dendrorhous or Candida albicans species.

In some aspects, the yeast cell is a Saccharomycete.

In some aspects, the yeast cell is a cell from Saccharomyces cerevisiaespecies.

The invention further provides a recombinant host cell comprisingexogenous nucleic acids comprising:

(a) a recombinant gene encoding a GGPPS;

(b) a recombinant gene encoding an ent-copalyl diphosphate synthase(CDPS) polypeptide;

(c) a recombinant gene encoding a kaurene oxidase (KO) polypeptide;

(d) a recombinant gene encoding a kaurene synthase (KS) polypeptide;

(e) a recombinant gene encoding a steviol synthase (KAH) polypeptide;

(f) a recombinant gene encoding a cytochrome P450 reductase (CPR)polypeptide; and/or

(g) a one or more recombinant genes encoding a one or more UGTpolypeptide;

wherein the cell produces Rebaudioside D, Rebaudioside M, RebaudiosideQ, or Rebaudioside I, di-glycosylated steviol glycoside (13-hydroxykaur-16-en-18-oic acid, [2-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester) or tri-glycosylated steviol glycoside (13-hydroxykaur-16-en-18-oic acid;[2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester).

In some aspects of said recombinant host cells, the GGPPS comprisesSynechococcus sp. GGPPS set forth in SEQ ID NO:24; the CDP polypeptidecomprises a Z. mays CDPS polypeptide set forth in SEQ ID NO:13, whereinthe polypeptide is lacking a chloroplast transit peptide; the KOpolypeptide comprises a KO polypeptide having 70% or greater identity tothe amino acid sequence of the S. rebaudiana KO polypeptide set forth inSEQ ID NO:25; the KS polypeptide comprises a KS polypeptide having 40%or greater identity to the amino acid sequence of the A. thaliana KSpolypeptide set forth in SEQ ID NO:21; the KAH polypeptide comprises aKAH polypeptide having 60% or greater identity to the S. rebaudiana KAHamino acid sequence set forth in SEQ ID NO:11; the CPR polypeptidecomprises a CPR polypeptide having 65% or greater identity to a S.rebaudiana CPR amino acid sequence set forth in SEQ ID NO:4, an A.thaliana CPR polypeptide of the amino acid sequence set forth in SEQ IDNO:9 or a combination thereof.

In some aspects, the cell produces Rebaudioside D or Rebaudioside M,wherein the UGT polypeptide is at least one UGT polypeptide that is:

a UGT85C2 polypeptide comprising a UGT85C2 polypeptide having 55% orgreater identity to the amino acid sequence set forth in SEQ ID NO:26;

a UGT74G1 polypeptide comprising a UGT74G1 polypeptide having 55% orgreater identity to the amino acid sequence set forth in SEQ ID NO:19;

a UGT76G1 polypeptide comprising a UGT76G1 polypeptide having 50% orgreater identity to the amino acid sequence set forth in SEQ ID NO:2;

a UGT91d2 polypeptide comprising a UGT91d2 polypeptide having 90% orgreater identity to the amino acid sequence set forth in SEQ ID NO:26 ora functional homolog thereof,

a UGT91d2e polypeptide having a substitution at residues 211 and 286 ofSEQ ID NO:15 or a combination thereof; or

a EUGT11 polypeptide comprising a EUGT11 polypeptide having 65% orgreater identity to the Os03g0702000 amino acid sequence set forth inSEQ ID NO:16.

In some aspects, the cell produces Rebaudioside Q, wherein the UGTpolypeptide is at least one UGT polypeptide that is:

a UGT85C2 polypeptide comprising a UGT85C2 polypeptide having 55% orgreater identity to the amino acid sequence set forth in SEQ ID NO:26; aUGT74G1 polypeptide comprising a UGT74G1 polypeptide having 55% orgreater identity to the amino acid sequence set forth in SEQ ID NO:19;or a UGT76G1 polypeptide comprising a UGT76G1 polypeptide having 50% orgreater identity to the amino acid sequence set forth in SEQ ID NO:2.

In some aspects, the cell produces Rebaudioside I, wherein the UGTpolypeptide is at least one UGT polypeptide that is:

a UGT85C2 polypeptide comprising a UGT85C2 polypeptide having 55% orgreater identity to the amino acid sequence set forth in SEQ ID NO:26; aUGT74G1 polypeptide comprising a UGT74G1 polypeptide having 55% orgreater identity to the amino acid sequence set forth in SEQ ID NO:19; aUGT76G1 polypeptide comprising a UGT76G1 polypeptide having 50% orgreater identity to the amino acid sequence set forth in SEQ ID NO:2; aUGT91d2 polypeptide comprising a UGT91d2 polypeptide having 90% orgreater identity to the amino acid sequence set forth in SEQ ID NO:26 ora functional homolog thereof, a UGT91d2e polypeptide having asubstitution at residues 211 and 286 of SEQ ID NO:15; or a combinationthereof.

In some aspects, the cell produces di-glycosylated steviol glycoside(13-hydroxy kaur-16-en-18-oic acid,[2-O-β-D-glucopyranosyl-β-D-glucopyranosyl] ester), wherein the UGTpolypeptide is at least one UGT polypeptide that is:

a UGT74G1 polypeptide comprising a UGT74G1 polypeptide having 55% orgreater identity to the amino acid sequence set forth in SEQ ID NO:19;or a EUGT11 polypeptide comprising a EUGT11 polypeptide having 65% orgreater identity to the Os03g0702000 amino acid sequence set forth inSEQ ID NO:16.

In some aspects, the cell produces tri-glycosylated steviol glycoside(13-hydroxy kaur-16-en-18-oic acid;[2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-3-D-glucopyranosyl]ester),wherein the UGT polypeptide is at least one UGT polypeptide that is:

a UGT76G1 polypeptide comprising a UGT76G1 polypeptide having 50% orgreater identity to the amino acid sequence set forth in SEQ ID NO:2; aUGT74G1 polypeptide comprising a UGT74G1 polypeptide having 55% orgreater identity to the amino acid sequence set forth in SEQ ID NO:19;or a EUGT11 polypeptide comprising a EUGT11 polypeptide having 65% orgreater identity to the Os03g0702000 amino acid sequence set forth inSEQ ID NO:16

In some aspects, the UGT76G1 polypeptide comprises one or more of theUGT76G1 polypeptide variants comprising: Q23G, Q23H, I26F, I26W, T146A,T146G, T146P, H155R, L257P, L257W, L257T, L257G, L257A, L257R, L257E,S283G and S283N of SEQ ID NO:2.

In some aspects, the UGT76G1 polypeptide comprises one or more of theUGT76G1 polypeptide variants comprising: T55K, T55E, S56A, Y128S, Y128E,H155L, H155R, Q198R, S285R, S285T, S253W, S253G, T284R, T284G, S285G,K337E, K337P and L379V of SEQ ID NO:2.

In some aspects, the recombinant host cell is a yeast cell, a plantcell, a mammalian cell, an insect cell, a fungal cell or a bacterialcell.

In some aspects, the yeast cell is a cell from Saccharomyces cerevisiae,Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbyagossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis,Hansenula polymorpha, Candida boidinii, Arxula adeninivorans,Xanthophyllomyces dendrorhous or Candida albicans species.

In some aspects, the yeast cell is a Saccharomycete.

In some aspects, the yeast cell is a cell from Saccharomyces cerevisiaespecies.

The invention further provides methods for producing Rebaudioside D byfermentation using a recombinant cell as disclosed herein.

The invention further provides methods for producing Rebaudioside M byfermentation using a recombinant cell as disclosed herein.

The invention further provides methods for producing Rebaudioside Q byfermentation using a recombinant cell as disclosed herein.

The invention further provides methods for producing Rebaudioside I byfermentation using a recombinant cell as disclosed herein.

The invention further provides methods for producing di-glycosylatedsteviol glycoside (13-hydroxy kaur-16-en-18-oic acid,[2-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester) by fermentation usinga recombinant cell as disclosed herein.

The invention further provides methods for producing tri-glycosylatedsteviol glycoside (13-hydroxy kaur-16-en-18-oic acid;[2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester) by fermentation using a recombinant cell as disclosed herein.

In some aspects, a cell for practicing the methods disclosed herein is ayeast cell, a plant cell, a mammalian cell, an insect cell, a fungalcell or a bacterial cell.

In some aspects, the yeast cell is a cell from Saccharomyces cerevisiae,Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbyagossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis,Hansenula polymorpha, Candida boidinii, Arxula adeninivorans,Xanthophyllomyces dendrorhous or Candida albicans species.

In some aspects, the yeast cell is a Saccharomycete.

In some aspects, the yeast cell is a cell from Saccharomyces cerevisiaespecies.

The invention further provides in vitro methods for producingRebaudioside D or Rebaudioside M, comprising:

(a) adding one or more of a UGT85C2 polypeptide comprising a UGT85C2polypeptide having 55% or greater identity to the amino acid sequenceset forth in SEQ ID NO:26; a UGT74G1 polypeptide comprising a UGT74G1polypeptide having 55% or greater identity to the amino acid sequenceset forth in SEQ ID NO:19; a UGT76G1 polypeptide comprising a UGT76G1polypeptide having 50% or greater identity to the amino acid sequenceset forth in SEQ ID NO:2; a UGT91d2 polypeptide comprising a UGT91d2polypeptide having 90% or greater identity to the amino acid sequenceset forth in SEQ ID NO:26 or a functional homolog thereof, a UGT91d2epolypeptide having a substitution at residues 211 and 286 of SEQ IDNO:15 or a combination thereof; a EUGT11 polypeptide comprising a EUGT11polypeptide having 65% or greater identity to the Os03g0702000 aminoacid sequence set forth in SEQ ID NO:16, and plant-derived or syntheticsteviol or steviol glycosides to the reaction mixture; and

(b) synthesizing Rebaudioside D or Rebaudioside M in the reactionmixture; and optionally

(c) isolating Rebaudioside D or Rebaudioside M in the reaction mixture.

The invention further provides in vitro methods for producingRebaudioside Q, comprising:

(a) adding one or more of a UGT85C2 polypeptide comprising a UGT85C2polypeptide having 55% or greater identity to the amino acid sequenceset forth in SEQ ID NO:26; a UGT74G1 polypeptide comprising a UGT74G1polypeptide having 55% or greater identity to the amino acid sequenceset forth in SEQ ID NO:19; a UGT76G1 polypeptide comprising a UGT76G1polypeptide having 50% or greater identity to the amino acid sequenceset forth in SEQ ID NO:2, and plant-derived or synthetic steviol orsteviol glycosides to the reaction mixture; and

(b) synthesizing Rebaudioside Q in the reaction mixture; and optionally

(c) isolating Rebaudioside Q in the reaction mixture.

The invention further provides in vitro methods for producingRebaudioside I, comprising:

(a) adding one or more of a UGT85C2 polypeptide comprising a UGT85C2polypeptide having 55% or greater identity to the amino acid sequenceset forth in SEQ ID NO:26; a UGT74G1 polypeptide comprising a UGT74G1polypeptide having 55% or greater identity to the amino acid sequenceset forth in SEQ ID NO:19; a UGT76G1 polypeptide comprising a UGT76G1polypeptide having 50% or greater identity to the amino acid sequenceset forth in SEQ ID NO:2; a UGT91d2 polypeptide comprising a UGT91d2polypeptide having 90% or greater identity to the amino acid sequenceset forth in SEQ ID NO:26 or a functional homolog thereof, a UGT91d2epolypeptide having a substitution at residues 211 and 286 of SEQ IDNO:15 or a combination thereof, and plant-derived or synthetic steviolor steviol glycosides to the reaction mixture; and

(b) synthesizing Rebaudioside I in the reaction mixture; and optionally

(c) isolating Rebaudioside I in the reaction mixture.

The invention further provides in vitro methods for producing adi-glycosylated steviol glycoside (13-hydroxy kaur-16-en-18-oic acid,[2-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester), comprising:

(a) adding one or more of a a UGT74G1 polypeptide comprising a UGT74G1polypeptide having 55% or greater identity to the amino acid sequenceset forth in SEQ ID NO:19; a EUGT11 polypeptide comprising a EUGT11polypeptide having 65% or greater identity to the Os03g0702000 aminoacid sequence set forth in SEQ ID NO:16, and plant-derived or syntheticsteviol or steviol glycosides to the reaction mixture; and

(b) synthesizing the di-glycosylated steviol glycoside (13-hydroxykaur-16-en-18-oic acid, [2-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester) in the reaction mixture; and optionally

(c) isolating di-glycosylated steviol glycoside (13-hydroxykaur-16-en-18-oic acid, [2-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester) in the reaction mixture.

The invention further provides in vitro methods for producing atri-glycosylated steviol glycoside (13-hydroxy kaur-16-en-18-oic acid;[2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester), comprising:

(a) adding one or more of a a UGT76G1 polypeptide comprising a UGT76G1polypeptide having 50% or greater identity to the amino acid sequenceset forth in SEQ ID NO:2; a UGT74G1 polypeptide comprising a UGT74G1polypeptide having 55% or greater identity to the amino acid sequenceset forth in SEQ ID NO:19; a EUGT11 polypeptide comprising a EUGT11polypeptide having 65% or greater identity to the Os03g0702000 aminoacid sequence set forth in SEQ ID NO:16, and plant-derived or syntheticsteviol or steviol glycosides to the reaction mixture; and

(b) synthesizing tri-glycosylated steviol glycoside (13-hydroxykaur-16-en-18-oic acid;[2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester) in the reaction mixture; and optionally

(c) isolating tri-glycosylated steviol glycoside (13-hydroxykaur-16-en-18-oic acid;[2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester) in the reaction mixture.

In some aspects, the UGT76G1 polypeptide for producing Rebaudioside Dcomprises one or more of the UGT76G1 polypeptide variants selected fromthe group consisting of: Q23G, Q23H, I26F, I26W, T146A, T146G, T146P,H155R, L257P, L257W, L257T, L257G, L257A, L257R, L257E, S283G and S283Nof SEQ ID NO:2.

In some aspects, the UGT76G1 polypeptide for producing Rebaudioside M,Rebaudioside Q, Rebaudioside I, di-glycosylated steviol glycoside andtri-glycosylated steviol glycoside comprises one or more of the UGT76G1polypeptide variants selected from the group consisting of: T55K, T55E,S56A, Y128S, Y128E, H155L, H155R, Q198R, S285R, S285T, S253W, S253G,T284R, T284G, S285G, K337E, K337P and L379V of SEQ ID NO:2.

In some aspects, the in vitro method disclosed is enzymatic in vitromethod or whole cell in vitro method.

In some aspects, a cell for practicing the methods disclosed herein is ayeast cell, a plant cell, a mammalian cell, an insect cell, a fungalcell or a bacterial cell.

In some aspects, the yeast cell is a cell from Saccharomyces cerevisiae,Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbyagossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis,Hansenula polymorpha, Candida boidinii, Arxula adeninivorans,Xanthophyllomyces dendrorhous or Candida albicans species.

In some aspects, the yeast cell is a Saccharomycete.

In some aspects, the yeast cell is a cell from Saccharomyces cerevisiaespecies.

The invention further provides Rebaudioside Q produced by the methodsdisclosed herein.

The invention further provides Rebaudioside I produced by the methodsdisclosed herein.

The invention further provides a di-glycosylated steviol glycoside(13-hydroxy kaur-16-en-18-oic acid,[2-O-β-D-glucopyranosyl-β-D-glucopyranosyl] ester) produced by themethods disclosed herein.

The invention further provides a tri-glycosylated steviol glycoside(13-hydroxy kaur-16-en-18-oic acid;[2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester) produced by the methods disclosed herein.

The invention further provides a UGT76G1 polypeptide for producingRebaudioside M, Rebaudioside Q, Rebaudioside I, di-glycosylated steviolglycoside (13-hydroxy kaur-16-en-18-oic acid,[2-O-β-D-glucopyranosyl-β-D-glucopyranosyl] ester) or tri-glycosylatedsteviol glycoside (13-hydroxy kaur-16-en-18-oic acid;[2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester), wherein the UGT76G1 polypeptide comprises a UGT76G1 polypeptideof SEQ ID NO:2 or one or more of the UGT76G1 polypeptide variantscomprising: T55K, T55E, S56A, Y128S, Y128E, H155L, H155R, Q198R, S285R,S285T, S253W, S253G, T284R, T284G, S285G, K337E, K337P and L379V of SEQID NO:2.

The invention further provides a UGT76G1 polypeptide for producingRebaudioside D, wherein the UGT76G1 polypeptide comprises a UGT76G1polypeptide of SEQ ID NO:2 or one or more of the UGT76G1 polypeptidevariants comprising: Q23G, Q23H, I26F, I26W, T146A, T146G, T146P, H155R,L257P, L257W, L257T, L257G, L257A, L257R, L257E, S283G and S283N of SEQID NO:2.

The invention further provides recombinant host cell comprising arecombinant gene encoding a UGT76G1 polypeptide, wherein the UGT76G1polypeptide comprises one or more of the UGT76G1 polypeptide variantscomprising: T55K, T55E, S56A, Y128S, Y128E, H155L, H155R, Q198R, S285R,S285T, S253W, S253G, T284R, T284G, S285G, K337E, K337P and L379V of SEQID NO:2.

In some aspects, the recombinant host cell as disclosed herein producesRebaudioside D.

The invention further provides a recombinant host cell comprising arecombinant gene encoding a UGT76G1 polypeptide, wherein the UGT76G1polypeptide comprises one or more of the UGT76G1 polypeptide variantscomprising: Q23G, Q23H, I26F, I26W, T146A, T146G, T146P, H155R, L257P,L257W, L257T, L257G, L257A, L257R, L257E, S283G and S283N of SEQ IDNO:2.

In some aspects, the recombinant host cell as disclosed herein producesRebaudioside M, Rebaudioside Q, Rebaudioside I, di-glycosylated steviolglycoside (13-hydroxy kaur-16-en-18-oic acid,[2-O-β-D-glucopyranosyl-β-D-glucopyranosyl] ester) or tri-glycosylatedsteviol glycoside (13-hydroxy kaur-16-en-18-oic acid;[2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester).

In some aspects, the recombinant host cell is a yeast cell, a plantcell, a mammalian cell, an insect cell, a fungal cell or a bacterialcell.

In some aspects, the yeast cell is a cell from Saccharomyces cerevisiae,Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbyagossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis,Hansenula polymorpha, Candida boidinii, Arxula adeninivorans,Xanthophyllomyces dendrorhous or Candida albicans species.

In some aspects, the yeast cell is a Saccharomycete.

In some aspects, the yeast cell is a cell from Saccharomyces cerevisiaespecies.

The invention further provides a composition comprising from about 1% toabout 99% w/w of Rebaudioside D, wherein the composition has a reducedlevel of Stevia-derived contaminants relative to a stevia extract. Incertain instances, the at least one of said contaminants is aplant-derived compound that inter alia contributes to off-flavors in thesteviol glycoside product.

In some aspects, the composition has less than 0.1% of Stevia-derivedcontaminants relative to a stevia extract. In certain instances, the atleast one of said contaminants is a plant-derived compound that interalia contributes to off-flavors in the steviol glycoside product.

The invention further provides a food product comprising the compositionas disclosed herein.

In some aspects, the food product is a beverage or a beverageconcentrate.

Any of the hosts described herein can be a microorganism (e.g., aSaccharomycete such as Saccharomyces cerevisiae, or Escherichia coli),or a plant or plant cell (e.g., a Stevia such as a Stevia rebaudiana orPhyscomitrella).

These and other features and advantages of the present invention will bemore fully understood from the following detailed description of theinvention taken together with the accompanying claims. It is noted thatthe scope of the claims is defined by the recitations therein and not bythe specific discussion of features and advantages set forth in thepresent description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of the embodiments of the presentinvention can be best understood when read in conjunction with thefollowing drawings, where like structure is indicated with likereference numerals and in which:

FIG. 1 shows the steviol glycoside glycosylation reactions and theenzymes by which they are catalyzed.

FIG. 2 shows the chemical structure of Rebaudioside M (RebM).

FIG. 3 shows the chemical structure of Rebaudioside D (RebD).

FIG. 4 shows biochemical pathway for the production of steviol.

FIG. 5 is a representative chromatogram of Liquid Chromatography-MassSpectrometry (LC-MS) analysis showing formation of a hexa-glycosylatedsteviol glycoside at 1.31 min retention time. The traces, from top tobottom, correspond to the m/z indicated in Table 12.

FIG. 6 is a schematic of the methods for isolating hexa-glycosylatedsteviol glycosides.

FIG. 7A is a representative chromatogram of the semi-purifiedhexa-glycosylated steviol glycoside after flash chromatography.

FIG. 7B is a representative mass spectra from a liquidchromatography-quadrupole time-of-flight (LC-QTOF) analysis of thesemi-purified hexa-glycosylated steviol glycoside after flashchromatography.

FIG. 8A is a chromatogram indicating compounds produced by fermentationof yeast strain EFSC 3044.

FIG. 8B is the NMR structure of the indicated di-glycosylated steviolglycoside (13-hydroxy kaur-16-en-18-oic acid,[2-O-β-D-glucopyranosyl-□β-D-glucopyranosyl] ester), an analog ofsteviol-1,2-bioside. The IUPAC name for di-glycosylated steviolglycoside is(2S,3R,4S,5S,6R)-4,5-dihydroxy-6-(hydroxymethyl)-3-{[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}oxan-2-yl(1R,4S,5R,9S,10R,13S)-13-hydroxy-5,9-dimethyl-14-methylidenetetracyclo[11.2.1.0{circumflexover ( )}{1,10}.0{circumflex over ( )}{4,9}]hexadecane-5-carboxylate.

FIG. 8C is the structure of the NMR structure of the indicatedtri-glycosylated steviol glycoside (13-hydroxy kaur-16-en-18-oic acid;[2-O-β-D-glucopyranosyl-3-O-□β-D-glucopyranosyl-β-D-glucopyranosyl]ester, an isomer of RebB. The IUPAC name for tri-glycosylated steviolglycoside is(2S,3R,4S,5R,6R)-5-hydroxy-6-(hydroxymethyl)-3,4-bis({[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy})oxan-2-yl(1R,4S,5R,9S,10R,13S)-13-hydroxy-5,9-dimethyl-14-methylidenetetracyclo[11.2.1.0{circumflexover ( )}{1,10}0.{circumflex over ( )}{4,9}]hexadecane-5-carboxylate.

FIG. 9 shows RebD production by the EFSC 3261 yeast strain. Fourfermentations of the EFSC 3261 yeast strain in minimal medium (MM) areshown.

FIG. 10 shows RebD and RebM production by the EFSC 3297 yeast strain.

FIG. 11 shows RebD, RebM, and RebA production by the EFSC 3841 yeaststrain.

FIG. 12 compares RebD/RebM produced with one or two copies of UGT76G1.

FIG. 13A shows the relative rates of consumption of RebD and productionof RebM by UGT76G1.

FIG. 13B shows the relative rates of consumption of RebE and productionof RebD and RebM by UGT76G1.

FIG. 14 shows the variance in the three homology models of UGT76G1.

FIG. 15A is a scatter-plot of production of RebD and RebM in 96 and 4×24deep-well plates.

FIG. 15B is a box-plot of RebD and RebM production in 96 and 4×24deep-well plates.

FIG. 15C is a box-plot of RebD/RebM production in 96 and 4×24 deep-wellplates.

FIG. 16 shows all data points of the initial UGT76G1 site saturationscreen with wild type production shown as black triangles.

FIG. 17 shows the top RebD and RebM producing colonies selected forfurther study.

FIG. 18 shows a rescreen of UGT76G1 RebD and RebM top producers (asshown in FIG. 17) run in triplicate showing the same trends as theinitial screen.

FIG. 19A shows the relative rates of consumption of Rubusoside andproduction of 1,3-stevioside (RebG) and Rebaudioside Q (“RebQ”) byUGT76G1.

FIG. 19B shows the relative rates of consumption of 1,2-stevioside andproduction of RebA by UGT76G1.

FIG. 19C shows the relative rates of consumption of 1,2-bioside andproduction of RebB by UGT76G1.

FIG. 20 shows chromatograms of 1,2-stevioside and RebA with or withoutUGT76G1 peaks indicating production of Rebl.

Skilled artisans will appreciate that elements in the Figures areillustrated for simplicity and clarity and have not necessarily beendrawn to scale. For example, the dimensions of some of the elements inthe Figures can be exaggerated relative to other elements to helpimprove understanding of the embodiment(s) of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

All publications, patents and patent applications cited herein arehereby expressly incorporated by reference for all purposes.

Methods well known to those skilled in the art can be used to constructgenetic expression constructs and recombinant cells according to thisinvention. These methods include in vitro recombinant DNA techniques,synthetic techniques, in vivo recombination techniques, and polymerasechain reaction (PCR) techniques. See, for example, techniques asdescribed in Maniatis et al., 1989, MOLECULAR CLONING: A LABORATORYMANUAL, Cold Spring Harbor Laboratory, New York; Ausubel et al., 1989,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates andWiley Interscience, New York, and PCR Protocols: A Guide to Methods andApplications (Innis et al., 1990, Academic Press, San Diego, Calif.).

Before describing the present invention in detail, a number of termswill be defined. As used herein, the singular forms “a”, “an”, and “the”include plural referents unless the context clearly dictates otherwise.For example, reference to a “nucleic acid” means one or more nucleicacids.

It is noted that terms like “preferably”, “commonly”, and “typically”are not utilized herein to limit the scope of the claimed invention orto imply that certain features are critical, essential, or evenimportant to the structure or function of the claimed invention. Rather,these terms are merely intended to highlight alternative or additionalfeatures that can or cannot be utilized in a particular embodiment ofthe present invention.

For the purposes of describing and defining the present invention it isnoted that the term “substantially” is utilized herein to represent theinherent degree of uncertainty that can be attributed to anyquantitative comparison, value, measurement, or other representation.The term “substantially” is also utilized herein to represent the degreeby which a quantitative representation can vary from a stated referencewithout resulting in a change in the basic function of the subjectmatter at issue.

As used herein, the terms “polynucleotide”, “nucleotide”,“oligonucleotide”, and “nucleic acid” can be used interchangeably torefer to nucleic acid comprising DNA, RNA, derivatives thereof, orcombinations thereof.

As used herein, the term “and/or” is utilized to describe multiplecomponents in combination or exclusive of one another. For example, “x,y, and/or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, andz,” “(x and y) or z,” or “x or y or z.” In some embodiments, “and/or” isused to refer to the exogenous nucleic acids that a recombinant cellcomprises, wherein a recombinant cell comprises one or more exogenousnucleic acids selected from a group. In some embodiments, “and/or” isused to refer to production of steviol glycosides, wherein one or moresteviol glycosides selected from a group are produced. In someembodiments, “and/or” is used to refer to production of steviolglycosides, wherein one or more steviol glycosides are produced throughone or more of the following steps: culturing a recombinant cell,synthesizing one or more steviol glycosides in a cell, and isolating oneor more steviol glycosides.

Highly-glycosylated steviol glycosides can be present in trace amountsin the Stevia plant, but at levels so low that extraction from the plantis impractical for use of such glycosides in food and beverage systems.See, Hellfritsch et al., J. Agric. Food Chem. 60: 6782-6793 (2012);DuBois G E, Stephenson R A., J Med Chem. January; 28:93-98 (1985); andUS Patent Publication 2011-0160311.

Typically, stevioside and Rebaudioside A are the primary compounds incommercially-produced stevia extracts. Stevioside is reported to have amore bitter and less sweet taste than Rebaudioside A. The composition ofstevia extract can vary from lot to lot depending on the soil andclimate in which the plants are grown. Depending upon the sourced plant,the climate conditions, and the extraction process, the amount ofRebaudioside A in commercial preparations is reported to vary from 20 to97% of the total steviol glycoside content.

Other steviol glycosides are present in varying amounts in steviaextracts. For example, Rebaudioside B is typically present at less than1-2%, whereas Rebaudioside C can be present at levels as high as 7-15%.Rebaudioside D is typically present in levels of 2% or less, andRebaudioside F is typically present in compositions at 3.5% or less ofthe total steviol glycosides. The amount of the minor steviolglycosides, including but not limited to Rebaudioside M, can affect theflavor profile of a Stevia extract.

In addition, Rebaudioside D and other higher glycosylated steviolglycosides are thought to be higher quality sweeteners than RebaudiosideA. As such, the recombinant hosts and methods described herein areparticularly useful for producing steviol glycoside compositions havingan increased amount of Rebaudioside D for use, for example, as anon-caloric sweetener with functional and sensory properties superior tothose of many high-potency sweeteners.

Rebaudioside M, a hexa-glycosylated steviol glycoside has been reportedto be present in the Stevia plant and has an IUPAC name of(2S,3R,4S,5R,6R)-5-hydroxy-6-(hydroxymethyl)-3,4-bis({[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy})oxan-2-yl(1R,5R,9S,13R)-13-{[(2S,3R,4S,5R,6R)-5-hydroxy-6-(hydroxymethyl)-3,4-bis({[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy})oxan-2-yl]oxy}-5,9-dimethyl-14-methylidenetetracyclo[11.2.1.01,10.04,9]hexadecane-5-carboxylate. See, Ohta et al., MassBank record: FU000341,FU000342, FU000343 (2010) and Ohta et al (J. Applied Glycosides,57(3):199-209, 2010). Rebaudioside M has been given a CAS number of1220616-44-3. See FIG. 2 for the structure of Rebaudioside M.

Rebaudioside D, a penta-glycosylated steviol glycoside, has also beenreported to be present in the Stevia plant and has an IUPAC name of4,5-dihydroxy-6-(hydroxymethyl)-3-{[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}oxan-2-yl13-{[5-hydroxy-6-(hydroxymethyl)-3,4-bis({[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy})oxan-2-yl]oxy}-5,9-dimethyl-14-methylidenetetracyclo[11.2.1.^(01,10).0^(4,9)]hexadecane-5-carboxylate.Rebaudioside D has been given a CAS number of 64849-39-4. See FIG. 3 forthe structure of Rebaudioside D.

Provided herein are recombinant hosts such as microorganisms thatexpress polypeptides useful for de novo biosynthesis of Rebaudioside Mor Rebaurdioside D. Hosts described herein express one or more uridine5′-diphospho (UDP) glycosyl transferases suitable for producing steviolglycosides. Expression of these biosynthetic polypeptides in variousmicrobial systems allows steviol glycosides to be produced in aconsistent, reproducible manner from energy and carbon sources such assugars, glycerol, CO₂, H₂, and sunlight. The proportion of each steviolglycoside produced by a recombinant host can be tailored byincorporating preselected biosynthetic enzymes into the hosts andexpressing them at appropriate levels, to produce a sweetenercomposition with a consistent taste profile. Furthermore, theconcentrations of steviol glycosides produced by recombinant hosts areexpected to be higher than the levels of steviol glycosides produced inthe Stevia plant, which improves the efficiency of the downstreampurification. Such sweetener compositions advantageously contain littleor no plant based contaminants, relative to the amount of contaminantspresent in Stevia extracts.

The practice of the methods and recombinant host cells as disclosed areprovided wherein at least one of the genes encoding a UGT85C2polypeptide; a UGT74G1 polypeptide; a UGT76G1 polypeptide; or a UGT91d2polypeptide is a recombinant gene, the particular recombinant gene(s)depending on the species or strain selected for use. Additional genes orbiosynthetic modules can be included in order to increase steviolglycoside yield, improve efficiency with which energy and carbon sourcesare converted to steviol and its glycosides, and/or to enhanceproductivity from the cell culture. As used herein, “biosyntheticmodules” refer to a collection of genes that are part of a commonbiosynthetic pathway and thus are often co-expressed in a recombinantorganism. As used herein, such additional biosynthetic modules includegenes involved in the synthesis of the terpenoid precursors, isopentenyldiphosphate and dimethylallyl diphosphate. Additional biosyntheticmodules include terpene synthase and terpene cyclase genes, such asgenes encoding geranylgeranyl diphosphate synthase and copalyldiphosphate synthase; these genes can be endogenous genes or recombinantgenes.

I. Steviol and Steviol Glycoside Biosynthesis Polypeptides A. SteviolBiosynthesis Polypeptides

The biochemical pathway to produce steviol involves formation of theprecursor, geranylgeranyl diphosphate (catalyzed by GGDPS), cyclizationto (-) copalyl diphosphate (catalyzed by CDPS), followed by formation of(-)-kaurene (catalyzed by KS), followed by oxidation (catalyzed by KO),and hydroxylation (catalyzed by KAH) to form steviol. See FIG. 4. Thus,conversion of geranylgeranyl diphosphate to steviol in a recombinantmicroorganism involves the expression of a gene encoding a kaurenesynthase (KS), a gene encoding a kaurene oxidase (KO), and a geneencoding a steviol synthetase (KAH). Steviol synthetase also is known askaurenoic acid 13-hydroxylase.

Suitable KS polypeptides are known. For example, suitable KS enzymesinclude those made by Stevia rebaudiana, Zea mays, Populus trichocarpa,and Arabidopsis thaliana. See, Table 2 and PCT Application Nos.PCT/US2012/050021 and PCT/US2011/038967, which are incorporated hereinby reference in their entirety. The nucleotide sequence encoding the A.thaliana KS polypeptide is set forth in SEQ ID NO:6, and the amino acidsequence of the A. thaliana KS polypeptide is set forth in SEQ ID NO:21.

TABLE 2 Kaurene synthase (KS) clones. Enzyme Source Accession ConstructOrganism gi Number Number Name Length (nts) Stevia 4959241 AAD34295MM-12 2355 rebaudiana (amino acid SEQ (nt SEQ ID NO: 103) ID NO: 27)Stevia 4959239 AAD34294 MM-13 2355 rebaudiana (amino acid SEQ (nt SEQ IDNO: 104) ID NO: 28) Zea mays 162458963 NP_001105097 MM-14 1773 (aminoacid SEQ (nt SEQ ID NO: 105) ID NO: 29) Populus 224098838 XP_002311286MM-15 2232 trichocarpa (amino acid SEQ (nt SEQ ID NO: 106) ID NO: 30)Arabidopsis 3056724 AF034774 EV-70 2358 thaliana (amino acid SEQ (nt SEQID NO: 31) ID NO: 32)

Suitable KO polypeptides are known. For example, suitable KO enzymesinclude those made by Stevia rebaudiana, Arabidopsis thaliana,Gibberella fujikoroi and Trametes versicolor. See, e.g., Table 3 and PCTApplication Nos. PCT/US2012/050021 and PCT/US2011/038967, which areincorporated herein by reference in their entirety.

TABLE 3 Kaurene oxidase (KO) clones. Enzyme Source Accession ConstructOrganism gi Number Number Name Length (nts) Stevia 76446107 ABA42921MM-18 1542 rebaudiana (amino acid SEQ (nt SEQ ID NO: 107) ID NO: 33)Arabidopsis 3342249 AAC39505 MM-19 1530 thaliana (amino acid SEQ (nt SEQID NO: 108) ID NO: 34) Gibberella 4127832 CAA76703 MM-20 1578 fujikoroi(amino acid SEQ (nt SEQ ID NO: 109) ID NO: 35) Trametes 14278967BAB59027 MM-21 1500 versicolor (amino acid SEQ (nt SEQ ID NO: 110) IDNO: 36)

Suitable KAH polypeptides are known. For example, suitable KAH enzymesinclude those made by Stevia rebaudiana, Arabidopsis thaliana, Vitisvinifera and Medicago trunculata. See, e.g., Table 4, PCT ApplicationNos. PCT/US2012/050021 and PCT/US2011/038967, U.S. Patent PublicationNos. 2008/0271205 and 2008/0064063, and Genbank Accession No. gi189098312 (SEQ ID NO: 37) and GenBank Accession ABD60225; G1:89242710(SEQ ID NO: 38), which are incorporated herein by reference in theirentirety. The steviol synthetase from A. thaliana is classified as aCYP714A2.

TABLE 4 Steviol synthetase (KAH) clones. Enzyme Source Accession PlasmidConstruct Organism gi Number Number Name Name Length (nts) Stevia *(amino acid SEQ pMUS35 MM-22 1578 rebaudiana ID NO: 43) (nt SEQ ID NO:111) Stevia 189418962 ACD93722 pMUS36 MM-23 1431 rebaudiana (amino acidSEQ (nt SEQ ID NO: 112) ID NO: 39) Arabidopsis 15238644 NP_197872 pMUS37MM-24 1578 thaliana (amino acid SEQ (nt SEQ ID NO: 113) ID NO: 40) Vitisvinifera 225458454 XP_002282091 pMUS38 MM-25 1590 (amino acid SEQ (ntSEQ ID NO: 114) ID NO: 41) Medicago 84514135 ABC59076 pMUS39 MM-26 1440trunculata (amino acid SEQ (nt SEQ ID NO: 115) ID NO: 42) * = Sequenceis identified with sequence identifier number 2 as shown in U.S. PatentPublication No. 2008-0064063.

In addition, a KAH polypeptide from Stevia rebaudiana that wasidentified as described in PCT Application No. PCT/US2012/050021, whichis incorporated herein by reference in its entirety, is particularlyuseful in a recombinant host. The nucleotide sequence encoding the S.rebaudiana KAH (SrKAHe1) is set forth in SEQ ID NO:91. A nucleotidesequence encoding the S. rebaudiana KAH that has been codon-optimizedfor expression in yeast is set forth in SEQ ID NO:8, and the amino acidsequence of the S. rebaudiana KAH polypeptide is set forth in SEQ IDNO:11. When expressed in S. cerevisiae, the S. rebaudiana KAH (SEQ IDNO:11) shows significantly higher steviol synthase activity as comparedto the A. thaliana ent-kaurenoic acid hydroxylase described by Yamaguchiet al. (U.S. Patent Publication No. 2008/0271205 A1) and other S.rebaudiana KAH enzymes described in U.S. Patent Publication No.2008/0064063 as well as the protein sequence deposited in GenBank asACD93722. The S. rebaudiana KAH polypeptide (SEQ ID NO:11) has less than20% identity to the KAH from U.S. Patent Publication No. 2008/0271205and less than 35% identity to the KAH from U.S. Patent Publication No.2008/0064063.

For example, the steviol synthase encoded by SrKAHe1 is activated by theS. cerevisiae CPR encoded by gene NCP1 (YHR042W). Greater activationlevels of the steviol synthase encoded by SrKAHe1 is observed when theA. thaliana CPR encoded by the gene ATR2 (SEQ ID NO:10) and the S.rebaudiana CPR encoded by the gene CPR8 (SEQ ID NO:5) are co-expressed.The amino acid sequence of the A. thaliana ATR2 is set forth in SEQ IDNO:9, and the amino acid sequence for S. rebaudiana CPR8 polypeptides isset forth in SEQ ID NO:4.

In some embodiments, a recombinant microorganism contains a recombinantgene encoding a KO, KS, and a KAH polypeptide. Such microorganisms alsotypically contain a recombinant gene encoding a cytochrome P450reductase (CPR) polypeptide, since certain combinations of KO and/or KAHpolypeptides require expression of an exogenous CPR polypeptide. Inparticular, the activity of a KO and/or a KAH polypeptide of plantorigin can be significantly increased by the inclusion of a recombinantgene encoding an exogenous CPR polypeptide. Suitable CPR polypeptidesare known. For example, suitable CPR enzymes include those made by S.rebaudiana and A. thaliana. See, e.g., Table 5 and PCT Application Nos.PCT/US2012/050021 and PCT/US2011/038967, which are incorporated hereinby reference in their entirety.

TABLE 5 Cytochrome P450 reductase (CPR) Clones. Enzyme Source AccessionPlasmid Construct Organism gi Number Number Name Name Length (nts)Stevia 93211213 ABB88839 pMUS40 MM-27 2133 rebaudiana (amino acid SEQ(nt SEQ ID NO: 116) ID NO: 44) Arabidopsis 15233853 NP_194183 pMUS41MM-28 2079 thaliana (amino acid SEQ (nt SEQ ID NO: 117) ID NO: 45)Giberella 32562989 CAE09055 pMUS42 MM-29 2142 fujikuroi (amino acid SEQ(nt SEQ ID NO: 118) ID NO: 46)

The yeast gene DPP1 and/or the yeast gene LPP1 can reduce the yield ofsteviol by converting the GGPP and FPP precursors by these enzymes.These genes can be disrupted or deleted such that the degradation offarnesyl pyrophosphate (FPP) to farnesol is reduced and the degradationof geranylgeranylpyrophosphate (GGPP) to geranylgeraniol (GGOH) isreduced. Alternatively, the promoter or enhancer elements of anendogenous gene encoding a phosphatase can be altered such that theexpression of their encoded proteins is altered. Homologousrecombination can be used to disrupt an endogenous gene. For example, a“gene replacement” vector can be constructed in such a way to include aselectable marker gene. The selectable marker gene can be operablylinked, at both 5′ and 3′ end, to portions of the gene of sufficientlength to mediate homologous recombination using methods known to thoseskilled in the art.

A selectable marker can be one of any number of genes that complementhost cell auxotrophy, provide antibiotic resistance, or result in acolor change. Linearized DNA fragments of the gene replacement vectorthen are introduced into the cells using methods well known in the art(see below). Integration of the linear fragments into the genome and thedisruption of the gene can be determined based on the selection markerand can be verified by, for example, Southern blot analysis. Subsequentto its use in selection, a selectable marker can be removed from thegenome of the host cell by, e.g., Cre-loxP systems (see, e.g., Gossen etal., 2002, Ann. Rev. Genetics 36:153-173 and U.S. ApplicationPublication No. 20060014264). Alternatively, a gene replacement vectorcan be constructed in such a way as to include a portion of the gene tobe disrupted, where the portion is devoid of any endogenous genepromoter sequence and encodes none, or an inactive fragment of, thecoding sequence of the gene.

An “inactive fragment” is a fragment of the gene that encodes a proteinhaving, e.g., less than about 10% (e.g., less than about 9%, less thanabout 8%, less than about 7%, less than about 6%, less than about 5%,less than about 4%, less than about 3%, less than about 2%, less thanabout 1%, or 0%) of the activity of the protein produced from thefull-length coding sequence of the gene. Such a portion of a gene isinserted in a vector in such a way that no known promoter sequence isoperably linked to the gene sequence, but that a stop codon and atranscription termination sequence are operably linked to the portion ofthe gene sequence. This vector can be subsequently linearized in theportion of the gene sequence and transformed into a cell. By way ofsingle homologous recombination, this linearized vector is thenintegrated in the endogenous counterpart of the gene with inactivationthereof.

Expression in a recombinant microorganism of these genes can result inthe conversion of geranylgeranyl diphosphate to steviol.

B. Steviol Glycoside Biosynthesis Polypeptides

Recombinant host cells are described herein that can convert steviol toa steviol glycoside. Such hosts (e.g., microorganisms) contains genesencoding one or more UDP Glycosyl Transferases, also known as UGTs. UGTstransfer a monosaccharide unit from an activated nucleotide sugar to anacceptor moiety, in this case, an —OH or —COOH moiety on steviol orsteviol derivative or an —OH moiety on a glucose already attached to thesteviol backbone. UGTs have been classified into families andsubfamilies based on sequence homology. See Li et al., 2001, J. Biol.Chem. 276:4338-4343.

Rubusoside Biosynthesis Polypeptides

Biosynthesis of rubusoside involves glycosylation of the 13-OH and the19-COOH of steviol. See FIG. 1. Conversion of steviol to rubusoside in arecombinant host such as a microorganism can be accomplished byexpression of gene(s) encoding UGTs 85C2 and 74G1, which transfer aglucose unit to the 13-OH or the 19-COOH, respectively, of steviol.

A suitable UGT85C2 functions as a uridine 5′-diphosphoglucosyl:steviol13-OH transferase, and a uridine5′-diphosphoglucosyl:steviol-19-O-glucoside 13-OH transferase. Exemplaryreactions for UGT85C2 include conversion of steviol and UDP-glucose toSteviol-13-O-glucoside or conversion of Steviol-19-O-glucoside andUDP-glucose to Rubusoside. See FIG. 1. Functional UGT85C2 polypeptidesalso can catalyze glucosyl transferase reactions that utilize steviolglycoside substrates other than steviol and steviol-19-O-glucoside.

A suitable UGT74G1 polypeptide functions as a uridine 5′-diphosphoglucosyl: steviol 19-COOH transferase and a uridine 5′-diphosphoglucosyl: steviol-13-O-glucoside 19-COOH transferase. Exemplaryreactions of 74G1 include conversion of steviol tosteviol-19-O-glucoside and conversion of steviol-13-O-glucoside toRubusoside. See FIG. 19 for these and other non-limiting examples ofUGT74G1 reactions. Functional UGT74G1 polypeptides also can catalyzeglycosyl transferase reactions that utilize steviol glycoside substratesother than steviol and steviol-13-O-glucoside, or that transfer sugarmoieties from donors other than uridine diphosphate glucose.

A recombinant microorganism expressing a functional UGT74G1 and afunctional UGT85C2 can make rubusoside and both steviol monosides (i.e.,steviol 13-O-monoglucoside and steviol 19-O-monoglucoside) when steviolis used as a feedstock in the medium. Typically, however, genes encodingUGT74G1 and UGT85C2 are recombinant genes that have been transformedinto a host (e.g., microorganism) that does not naturally possess them.

As used herein, the term “recombinant host” is intended to refer to(including but not limited to) a host cell, the genome of which has beenaugmented by at least one incorporated DNA sequence; extrachromosomalexamples, like plasmids in bacteria and episomes comprising the 2-microncircle in yeast. Such DNA sequences include but are not limited to genesthat are not naturally present, DNA sequences that are not normallytranscribed into RNA or translated into a protein (“expressed”), andother genes or DNA sequences which one desires to introduce into thenon-recombinant host. It will be appreciated that typically the genomeof a recombinant host described herein is augmented through the stableintroduction of one or more recombinant genes. Generally, the introducedDNA is not originally resident in the host that is the recipient of theDNA, but it is within the scope of the invention to isolate a DNAsegment from a given host, and to subsequently introduce one or moreadditional copies of that DNA into the same host, e.g., to enhanceproduction of the product of a gene or alter the expression pattern of agene. In some instances, the introduced DNA will modify or even replacean endogenous gene or DNA sequence by, e.g., homologous recombination orsite-directed mutagenesis. Suitable recombinant hosts includemicroorganisms, mammalian cells, insect cells, fungal cells, plantcells, and plants.

The term “recombinant gene” refers to a gene or DNA sequence that isintroduced into a recipient host, regardless of whether the same or asimilar gene or DNA sequence can already be present in such a host.“Introduced,” or “augmented” in this context, is known in the art tomean introduced or augmented by the hand of man. Thus, a recombinantgene can be a DNA sequence from another species, or can be a DNAsequence that originated from or is present in the same species, but hasbeen incorporated into a host by recombinant methods to form arecombinant host. It will be appreciated that a recombinant gene that isintroduced into a host can be identical to a DNA sequence that isnormally present in the host being transformed, and is introduced toprovide one or more additional copies of the DNA to thereby permitoverexpression or modified (including but not limited to regulated orinducible) expression of the gene product of that DNA.

Suitable UGT74G1 and UGT85C2 polypeptides include those made by S.rebaudiana. Genes encoding functional UGT74G1 and UGT85C2 polypeptidesfrom Stevia are reported in Richman et al., 2005, Plant J. 41: 56-67.Amino acid sequences of S. rebaudiana UGT74G1 (SEQ ID NO: 19) andUGT85C2 (SEQ ID NO: 26) polypeptides are set forth in SEQ ID NOs: 1 and3, respectively, of PCT Application No. PCT/US2012/050021, as arenucleotide sequences that encode UGT74G1 and UGT85C2 that have beenoptimized for expression in yeast and DNA 2.0 codon-optimized sequencefor UGTs 85C2, 91D2e, 74G1 and 76G1. The Gene Art codon optimizednucleotide sequence encoding a S. rebaudiana UGT85C2 is set forth in SEQID NO:3. See also, the UGT85C2 and UGT74G1 variants described below inthe “Functional Homolog” section. For example, a UGT85C2 polypeptide cancontain substitutions at any one of the positions 65, 71, 270, 289, and389 (e.g., A65S, E71Q, T270M, Q289H, and A389V) or a combinationthereof.

In some embodiments, the recombinant host is a microorganism.Recombinant microorganism can be grown on media containing steviol inorder to produce rubusoside. In other embodiments, however, therecombinant microorganism expresses genes involved in steviolbiosynthesis, e.g., a CDPS gene, a KS gene, a KO gene, and a KAH gene.Suitable CDPS polypeptides are known. For example, suitable CDPS enzymesinclude those made by S. rebaudiana, Streptomyces clavuligerus,Bradyrhizobium japonicum, Zea mays, and Arabidopsis sp. See, e.g., Table6 and PCT Application Nos. PCT/US2012/050021 and PCT/US2011/038967,which are incorporated herein by reference in their entirety.

In some embodiments, CDPS polypeptides that lack a chloroplast transitpeptide at the amino terminus of the unmodified polypeptide can be used.For example, the first 150 nucleotides from the 5′ end of the Zea maysCDPS coding sequence (SEQ ID NO:12) can be removed, the truncatednucleotide sequence is shown in SEQ ID NO:133. Doing so removes theamino terminal 50 residues of the amino acid sequence shown in SEQ IDNO:13, which encode a chloroplast transit peptide; the truncated aminoacid sequence is shown in SEQ ID NO:134. The truncated CDPS gene can befitted with a new ATG translation start site and operably linked to apromoter, typically a constitutive or highly expressing promoter. When aplurality of copies, including but not limited to, one copy, two copiesor three copies of the truncated coding sequence are introduced into amicroorganism, expression of the CDPS polypeptide from the promoterresults in an increased carbon flux towards ent-kaurene biosynthesis.

TABLE 6 CDPS Clones. Enzyme Source Accession Plasmid Construct Organismgi Number Number Name Name Length (nts) Stevia 2642661 AAB87091 pMUS22MM-9 2364 rebaudiana (amino acid SEQ (nt SEQ ID NO: 119) ID NO: 48)Streptomyces 197705855 EDY51667 pMUS23 MM-10 1584 clavuligerus (aminoacid SEQ (nt SEQ ID NO: 120) ID NO: 49) Bradyrhizobium 529968 AAC28895.1pMUS24 MM-11 1551 japonicum (amino acid SEQ (nt SEQ ID NO: 121) ID NO:50) Zea mays 50082774 AY562490 EV65 2484 (amino acid SEQ (nt SEQ ID NO:51) ID NO: 52) Arabidopsis 18412041 NM_116512 EV64 2409 thaliana (SEQ IDNO: 54) (nt SEQ ID NO: 53)

CDPS-KS bifunctional proteins also can be used. Nucleotide sequencesencoding the CDPS-KS bifunctional enzymes shown in Table 7 were modifiedfor expression in yeast (see PCT Application No. PCT/US2012/050021,which is incorporated herein by reference in its entirety). Abifunctional enzyme from Gibberella fujikuroi also can be used.

TABLE 7 CDPS-KS Clones. Enzyme Source Accession Construct Organism giNumber Number Name Length (nts) Phomopsis 186704306 BAG30962 MM-16 2952amygdali (amino acid SEQ (nt SEQ ID NO: 122) ID NO: 55) Physcomitrella146325986 BAF61135 MM-17 2646 patens (amino acid SEQ (nt SEQ ID NO: 123)ID NO: 56) Gibberella 62900107 Q9UVY5.1 2859 fujikuroi (amino acid SEQ(nt SEQ ID NO: 124) ID NO: 57)

Thus, a microorganism containing a CDPS gene, a KS gene, a KO gene and aKAH gene in addition to a UGT74G1 and a UGT85C2 gene is capable ofproducing both steviol monosides and rubusoside without the necessityfor using steviol as a feedstock.

In some embodiments, the recombinant microorganism further expresses arecombinant gene encoding a geranylgeranyl diphosphate synthase (GGPPS).Suitable GGPPS polypeptides are known. For example, suitable GGPPSenzymes include those made by S. rebaudiana, Gibberella fujikuroi, Musmusculus, Thalassiosira pseudonana, Streptomyces clavuligerus,Sulfulobus acidocaldarius, Synechococcus sp. and A. thaliana. See, Table8 and PCT Application Nos. PCT/US2012/050021 and PCT/US2011/038967,which are incorporated herein by reference in their entirety.

TABLE 8 GGPPS Clones. Enzyme Source Accession Plasmid Construct Organismgi Number Number Name Name Length (nts) Stevia 90289577 ABD92926 pMUS14MM-1 1086 rebaudiana (amino acid SEQ (nt SEQ ID NO: 125) ID NO: 58)Gibberella 3549881 CAA75568 pMUS15 MM-2 1029 fujikuroi (amino acid SEQ(nt SEQ ID NO: 126) ID NO: 59) Mus musculus 47124116 AAH69913 pMUS16MM-3 903 (amino acid SEQ (nt SEQ ID NO: 127) ID NO: 60) Thalassiosira223997332 XP_002288339 pMUS17 MM-4 1020 pseudonana (amino acid SEQ (ntSEQ ID NO: 128) ID NO: 61) Streptomyces 254389342 ZP_05004570 pMUS18MM-5 1068 clavuligerus (amino acid SEQ (nt SEQ ID NO: 129) ID NO: 62)Sulfulobus 506371 BAA43200 pMUS19 MM-6 993 acidocaldarius (amino acidSEQ (nt SEQ ID NO: 130) ID NO: 63) Synechococcus 86553638 ABC98596pMUS20 MM-7 894 sp. (amino acid SEQ (nt SEQ ID NO: 131) ID NO: 64)Arabidopsis 15234534 NP_195399 pMUS21 MM-8 1113 thaliana (amino acid SEQ(nt SEQ ID NO: 132) ID NO: 63)

In some aspects, the KAH gene encoding the KAH polypeptide set forth inSEQ ID NO:11, comprising a recombinant cell of the invention isoverexpressed. In some aspects, the KAH gene can be present in(including but not limited to) one, two or three copies. In someaspects, the KS gene encoding the KS polypeptide, set forth in SEQ IDNO:21, comprising a recombinant cell of the invention is overexpressed.In some aspects, the KS gene can be present in (including but notlimited to) one, two or three copies.

In some embodiments, the recombinant microorganism further can expressrecombinant genes involved in diterpene biosynthesis or production ofterpenoid precursors, e.g., genes in the methylerythritol 4-phosphate(MEP) pathway or genes in the mevalonate (MEV) pathway discussed below,have reduced phosphatase activity, and/or express a sucrose synthase(SUS) as discussed herein.

Rebaudioside a, Rebaudioside D, and Rebaudioside E BiosynthesisPolypeptides

Biosynthesis of Rebaudioside A involves glucosylation of the aglyconesteviol. Specifically, Rebaudioside A can be formed by glucosylation ofthe 13-OH of steviol which forms the 13-O-steviolmonoside, glucosylationof the C-2′ of the 13-O-glucose of steviolmonoside which formssteviol-1,2-bioside, glucosylation of the C-19 carboxyl ofsteviol-1,2-bioside which forms stevioside, and glucosylation of theC-3′ of the C-13-O-glucose of stevioside to produce Reb A. The order inwhich each glucosylation reaction occurs can vary. See FIG. 1.

Biosynthesis of Rebaudioside E and/or Rebaudioside D involvesglucosylation of the aglycone steviol. Specifically, Rebaudioside E canbe formed by glucosylation of the 13-OH of steviol which formssteviol-13-O-glucoside, glucosylation of the C-2′ of the 13-O-glucose ofsteviol-13-O-glucoside which forms the steviol-1,2-bioside,glucosylation of the C-19 carboxyl of the 1,2-bioside to form1,2-stevioside, and glucosylation of the C-2′ of the 19-O-glucose of the1,2-stevioside to form Rebaudioside E. Rebaudioside D can be formed byglucosylation of the C-3′ of the C-13-O-glucose of Rebaudioside E. Theorder in which each glycosylation reaction occurs can vary. For example,the glucosylation of the C-2′ of the 19-O-glucose can be the last stepin the pathway, wherein Rebaudioside A is an intermediate in thepathway. See FIG. 1.

Rebaudioside M Polypeptides

As provided herein, conversion of steviol to Rebaudioside M in arecombinant host can be accomplished by expressing combinations of thefollowing functional UGTs: 91D2, EUGT11, 74G1, 85C2, and 76G1. SeeFIG. 1. It is particularly useful to express EUGT11 at high levels usinga high copy number plasmid, or using a strong promoter, or multipleintegrated copies of the gene, or episome under selection for high copynumber of the gene. Thus, a recombinant microorganism expressingcombinations of these UGTs can make Rebaudioside A (85C2; 76G1; 74G1;91D2e), Rebaudioside D (85C2; 76G1; 74G1; 91D2e; EUGT11), Rebaudioside E(85C2; 74G1; 91D2e; EUGT11), or Rebaudioside M (85C2; 76G1; 74G1; 91D2e;EUGT11). See FIG. 1. Typically, one or more of these genes arerecombinant genes that have been transformed into a microorganism thatdoes not naturally possess them. It has also been discovered that UGTsdesignated herein as SM12UGT can be substituted for UGT91 D2.

Targeted production of individual Rebaudiosides can be accomplished bydifferential copy numbers of the UGT-encoding genes (see FIG. 1) in therecombinant cell, differential promoter strengths, and/or by utilizingmutants with increased specificity/activity towards the product ofinterest. For example, low levels of Rebaudioside D, E, and M will beformed if EUGT11 is expressed at low levels in comparison to the otherUGTs, which would favor Rebaudioside A formation. High levels of EUGT11expression result in production of more 19-O 1,2 diglucoside that canserve as substrate for UGT76G1 to form Rebaudioside M. In certainadvantageous embodiments, additional copies or mutant versions ofUGT76G1 in recombinant cells of the invention can improve the rate ofRebaudioside M formation from Rebaudioside D.

In some embodiments, UGT76G1 catalyzes glycosylation of steviol andsteviol glycosides at the 19-O position. Thus, in some embodiments, oneor more of RebM, RebQ, Rebl, di-glycosylated steviol glycoside(13-hydroxy kaur-16-en-18-oic acid,[2-O-β-D-glucopyranosyl-β-D-glucopyranosyl] ester), or tri-glycosylatedsteviol glycoside ((13-hydroxy kaur-16-en-18-oic acid;[2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl]ester)are produced in a recombinant host expressing a recombinant geneencoding a UGT76G1 polypeptide, through bioconversion, or throughcatalysis by UGT76G1 in vitro. In some embodiments, UGT76G1 catalyzesthe glycosylation of steviol and steviol glycosides at the 13-O positionand preferentially glycosylates steviol glycoside substrates that are1,2-di-glycosylated at the 13-O position or mono-glycosylated at the13-O position. In some embodiments, UGT76G1 does not show a preferencefor the glycosylation state of the 19-O position.

In some aspects, a recombinant host cell of the invention comprises thegene encoding the UGT76G1 polypeptide set forth in SEQ ID NO:2. In someaspects, the gene encoding the UGT76G1 polypeptide set forth in SEQ IDNO:2, comprising a recombinant cell of the invention is overexpressed.In some aspects, the gene can be present in (including but not limitedto) two or three copies.

In some embodiments, the gene encoding the UGT76G1 polypeptide set forthin SEQ ID NO:2 is present in one copy. As shown in FIG. 12 (Example 9),a lower copy number (one copy) of the gene encoding the UGT76G1polypeptide results in lower UGT76G1 expression and increases theRebaudioside D/Rebaudioside M ratio.

In some embodiments, less than five (e.g., one, two, three, or four)UGTs are expressed in a host. For example, a recombinant microorganismexpressing a functional EUGT11 can make Rebaudioside D when RebaudiosideA is used as a feedstock. A recombinant microorganism expressing afunctional UGT76G1 can make Rebaudioside M when Rebaudioside D orRebaudioside E is used as a feedstock. Rebaudioside M can be formed fromeither Rebaudioside D or Rebaudioside E by glucosylation of the C-3′ ofthe 19-O-glucose of Rebaudioside D or Rebuadioside E; in the case ofRebaudioside E a second glucosylation is required, of the 13-O-glucoseto produce Rebaudioside M.

A recombinant microorganism expressing EUGT11, 74G1 or 76G1, and 91D2can make Rebaudioside D or Rebaudioside M when rubusoside or1,2-stevioside is used as a feedstock. As another alternative, arecombinant microorganism expressing EUGT11, 74G1, 76G1, and 91D2 canmake Rebaudioside D or Rebaudioside M when the monoside,steviol-13-O-glucoside are added to the medium. Similarly, conversion ofsteviol-19-O-glucoside to Rebaudioside D in a recombinant microorganismcan be accomplished by expressing in the cell genes encoding UGTsEUGT11, 85C2, 76G1, and 91D2e, when fed steviol-19-O-glucoside.

Suitable UGT74G1 and UGT85C2 polypeptides include those discussed above.A suitable UGT76G1 adds a glucose moiety to the C-3′ of theC-13-O-glucose of the acceptor molecule, a steviol 1,2 glycoside.UGT76G1 functions, for example, as a uridine 5′-diphospho glucosyl:steviol 13-O-1,2 glucoside C-3′ glucosyl transferase and a uridine5′-diphospho glucosyl: steviol-19-O-glucose, 13-O-1,2 bioside C-3′glucosyl transferase. Functional UGT76G1 polypeptides can also catalyzeglucosyl transferase reactions that utilize steviol glycoside substratesthat contain sugars other than glucose, e.g., steviol rhamnosides andsteviol xylosides. See, FIG. 1. Suitable UGT76G1 polypeptides includethose made by S. rebaudiana and reported in Richman et al., 2005, PlantJ. 41: 56-67. A nucleotide sequence encoding the S. rebaudiana UGT76G1polypeptide optimized for expression in yeast is set forth in SEQ IDNO:14. See also the UGT76G1 variants set forth in the “FunctionalHomolog” section.

A suitable EUGT11 or UGT91D2 polypeptide functions as a uridine5′-diphospho glucosyl: steviol-13-O-glucoside transferase (also referredto as a steviol-13-monoglucoside 1,2-glucosylase), transferring aglucose moiety to the C-2′ of the 13-O-glucose of the acceptor molecule,steviol-13-O-glucoside.

A suitable EUGT11 or UGT91D2 polypeptide also functions as a uridine5′-diphospho glucosyl: rubusoside transferase transferring a glucosemoiety to the C-2′ of the 13-O-glucose of the acceptor molecule,rubusoside, to produce stevioside. EUGT11 polypeptides also can transfera glucose moiety to the C-2′ of the 19-O-glucose of the acceptormolecule, rubusoside, to produce a 19-O-1,2-diglycosylated rubusoside(see FIG. 1).

Functional EUGT11 or UGT91D2 polypeptides also can catalyze reactionsthat utilize steviol glycoside substrates other thansteviol-13-O-glucoside and rubusoside. For example, a functional EUGT11polypeptide can utilize stevioside as a substrate, transferring aglucose moiety to the C-2′ of the 19-O-glucose residue to produceRebaudioside E (see FIG. 1). Functional EUGT11 and UGT91D2 polypeptidescan also utilize Rebaudioside A as a substrate, transferring a glucosemoiety to the C-2′ of the 19-O-glucose residue of Rebaudioside A toproduce Rebaudioside D. EUGT11 can convert Rebaudioside A toRebaudioside D at a rate that is least 20 times faster (e.g., as least25 times or at least 30 times faster) than the corresponding rate ofwildtype UGT91D2e (SEQ ID NO: 15) when the reactions are performed undersimilar conditions, i.e., similar time, temperature, purity, andsubstrate concentration. As such, EUGT11 produces greater amounts ofRebD than UGT91D2e under similar conditions in cells or in vitro, underconditions where the temperature-sensitive EUGT11 is stable.

In addition, a functional EUGT11 exhibits significant C-2′19-O-diglycosylation activity with rubusoside or stevioside assubstrates, whereas UGT91D2e has no detectable diglycosylation activitywith these substrates under some conditions. Thus, a functional EUGT11can be distinguished from UGT91D2e by the differences in steviolglycoside substrate-specificity.

A functional EUGT11 or UGT91 D2 polypeptide does not transfer a glucosemoiety to steviol compounds having a 1,3-bound glucose at the C-13position, i.e., transfer of a glucose moiety to steviol-1,3-bioside and1,3-stevioside (RebG) does not occur.

Functional EUGT11 and UGT91D2 polypeptides can transfer sugar moietiesfrom donors other than uridine diphosphate glucose. For example, afunctional EUGT11 or UGT91D2 polypeptide can act as a uridine5′-diphospho D-xylosyl: steviol-13-O-glucoside transferase, transferringa xylose moiety to the C-2′ of the 13-O-glucose of the acceptormolecule, steviol-13-O-glucoside. As another example, a functionalEUGT11 or UGT91D2 polypeptide can act as a uridine 5′-diphosphoL-rhamnosyl: steviol-13-O-glucoside transferase, transferring a rhamnosemoiety to the C-2′ of the 13-O-glucose of the acceptor molecule,steviol-13-O-glucoside.

Suitable EUGT11 polypeptides are described herein and can include theEUGT11 polypeptide from Oryza sativa (GenBank Accession No. AC133334;SEQ ID NO:16). For example, an EUGT11 polypeptide can have an amino acidsequence with at least 70% sequence identity (e.g., at least 75, 80, 85,90, 95, 96, 97, 98, or 99% sequence identity) to the amino acid sequenceset forth in SEQ ID NO:16. The nucleotide sequence encoding the aminoacid sequence of EUGT11 also is set forth in SEQ ID NO:17, as is a codonoptimized nucleotide sequence for expression in yeast (SEQ ID NO:18).

Suitable functional UGT91D2 polypeptides include, e.g., the polypeptidesdesignated UGT91D2e and UGT91D2m and functional homologs as describedherein. The amino acid sequence of an exemplary UGT91D2e polypeptidefrom S. rebaudiana is set forth in SEQ ID NO:15, as is the nucleotidesequence encoding the UGT91D2e polypeptide that has been codon optimizedfor expression in yeast (SEQ ID NO:89). The amino acid sequences ofexemplary UGT91D2m (SEQ ID NO:86) polypeptides from S. rebaudiana areset forth as SEQ ID NO: 10 in PCT Application No. PCT/US2012/050021,which is incorporated herein by reference in its entirety. In addition,UGT91D2 variants containing a substitution at amino acid residues 206,207, and 343 can be used. For example, the amino acid sequence havingG206R, Y207C, and W343R mutations with respect to wild-type UGT91D2e canbe used. In addition, a UGT91D2 variant containing substitutions atamino acid residues 211 and 286 can be used. For example, a UGT91D2variant can include a substitution of a methionine for leucine atposition 211 and a substitution of an alanine for valine at position 286(referred to as UGT91D2e-b). These variants, L211M and V286A, arevariants of SEQ ID NO: 5 from PCT/US2012/050021, which is disclosedherein as SEQ ID NO: 66. Additional variants can include variants(except T144S, M152L, L213F, S364P, and G384C variants) described inTable 12 and Example 11 of the PCT/US2012/050021, which is incorporatedherein by reference in its entirety.

As indicated above, UGTs designated herein as SM12UGT can be substitutedfor UGT91D2. Suitable functional SM12UGT polypeptides include those madeby Ipomoea purpurea (Japanese morning glory) and described in Morita etal., 2005, Plant J. 42, 353-363. The amino acid sequence encoding the I.purpurea IP3GGT (SEQ ID NO: 67) (which is set forth in PCT ApplicationNo. PCT/US2012/050021, which is incorporated herein by reference in itsentirety) as a nucleotide sequence (SEQ ID NO: 68) that encodes thepolypeptide and that has been codon optimized for expression in yeast.Another suitable SM12UGT polypeptide is a UGT94B1 polypeptide having anR25S mutation (Bp94B1 polypeptide). See Osmani et al., 2008, Plant Phys.148: 1295-1308 and Sawada et al., 2005, J. Biol. Chem. 280:899-906. Theamino acid sequence of the Bellis perennis (red daisy) UGT94B1 (SEQ IDNO: 69) and the nucleotide sequence that has been codon optimized forexpression in yeast (SEQ ID NO: 70) are set forth in PCT Application No.PCT/US2012/050021, which is incorporated herein by reference in itsentirety.

In some embodiments, the recombinant microorganism is grown on mediacontaining steviol-13-O-glucoside or steviol-19-O-glucoside in order toproduce Rebaudioside M. In such embodiments, the microorganism containsand expresses genes encoding a functional EUGT11, a functional UGT74G1,a functional UGT85C2, a functional UGT76G1, and a functional UGT91D2,and is capable of accumulating Rebaudioside A, Rebaudioside D,Rebaudioside M or a combination thereof, depending on the relativelevels of UDP-glycosyl transferase activities, when steviol, one or bothof the steviolmonosides, or rubusoside is used as feedstock.

In other embodiments, the recombinant microorganism is grown on mediacontaining rubusoside in order to produce Rebaudioside A, D, or M. Insuch embodiments, the microorganism contains and expresses genesencoding a functional EUGT11, a functional UGT76G1, and a functionalUGT91D2, and is capable of producing Rebaudioside A, D, M or acombination thereof, depending on the relative levels of UDP-glycosyltransferase activities, when rubusoside is used as feedstock.

In other embodiments the recombinant microorganism expresses genesinvolved in steviol biosynthesis, e.g., a CDPS gene, a KS gene, a KOgene and/or a KAH gene. Thus, for example, a microorganism containing aCDPS gene, a KS gene, a KO gene and a KAH gene, in addition to a EUGT11,a UGT74G1, a UGT85C2, a UGT76G1, and a functional UGT91D2 (e.g.,UGT91D2e), is capable of producing Rebaudioside A, D, E, and/or Mwithout the necessity of including steviol in the culture media.

In some embodiments, the recombinant host further contains and expressesa recombinant GGPPS gene in order to provide increased levels of thediterpene precursor geranylgeranyl diphosphate, for increased fluxthrough the steviol biosynthetic pathway.

In some embodiments, the recombinant host further contains a constructto silence expression of non-steviol pathways consuming geranylgeranyldiphosphate, ent-Kaurenoic acid or farnesyl pyrophosphate, therebyproviding increased flux through the steviol and steviol glycosidesbiosynthetic pathways. As discussed below, flux to sterol productionpathways such as ergosterol can be reduced by downregulation of the ERG9gene. In cells that produce gibberellins, gibberellin synthesis can bedownregulated to increase flux of ent-kaurenoic acid to steviol. Incarotenoid-producing organisms, flux to steviol can be increased bydownregulation of one or more carotenoid biosynthetic genes. In someembodiments, the recombinant microorganism further can expressrecombinant genes involved in diterpene biosynthesis or production ofterpenoid precursors, e.g., genes in the MEP or MEV pathways discussedbelow, have reduced phosphatase activity, and/or express a SUS asdiscussed herein.

One with skill in the art will recognize that by modulating relativeexpression levels of different UGT genes, a recombinant host can betailored to specifically produce steviol glycoside products in a desiredproportion. Transcriptional regulation of steviol biosynthesis genes andsteviol glycoside biosynthesis genes can be achieved by a combination oftranscriptional activation and repression using techniques known tothose in the art. For in vitro reactions, one with skill in the art willrecognize that addition of different levels of UGT enzymes incombination or under conditions which impact the relative activities ofthe different UGTS in combination will direct synthesis towards adesired proportion of each steviol glycoside. One with skill in the artwill recognize that a higher proportion of Rebaudioside D or M, or moreefficient conversion to Rebaudioside D or M can be obtained with adiglycosylation enzyme that has a higher activity for the 19-O-glucosidereaction as compared to the 13-O-glucoside reaction (substratesRebaudioside A and stevioside).

In some embodiments, a recombinant host such as a microorganism producesRebaudioside M-enriched steviol glycoside compositions that have greaterthan at least 3% Rebaudioside M by weight total steviol glycosides,e.g., at least 4% Rebaudioside M, at least 5% Rebaudioside M, at least10-20% Rebaudioside M, at least 20-30% Rebaudioside M, at least 30-40%Rebaudioside M, at least 40-50% Rebaudioside M, at least 50-60%Rebaudioside M, at least 60-70% Rebaudioside M, or at least 70-80%Rebaudioside M. Other steviol glycosides present can include thosedepicted in FIG. 1 such as steviol monosides, steviol glucobiosides,Rebaudioside A, Rebaudioside D, Rebaudioside E, and stevioside. In someembodiments, the Rebaudioside M-enriched composition produced by thehost (e.g., microorganism) can be further purified and the RebaudiosideM so purified can then be mixed with other steviol glycosides, flavors,or sweeteners to obtain a desired flavor system or sweeteningcomposition. For instance, a Rebaudioside M-enriched compositionproduced by a recombinant host can be combined with a Rebaudioside A, C,D, or E-enriched composition produced by a different recombinant host,with Rebaudioside A, C, D, or E purified from a Stevia extract, or withRebaudioside A, C, D, or E produced in vitro.

In some embodiments, Rebaudioside M can be produced using in vitromethods while supplying the appropriate UDP-sugar and/or a cell-freesystem for regeneration of UDP-sugars. In some embodiments, sucrose anda sucrose synthase can be provided in the reaction vessel in order toregenerate UDP-glucose from the UDP generated during glycosylationreactions. The sucrose synthase can be from any suitable organism. Forexample, a sucrose synthase coding sequence from A. thaliana, S.rebaudiana, or Coffea arabica can be cloned into an expression plasmidunder control of a suitable promoter, and expressed in a host such as amicroorganism or a plant.

Conversions requiring multiple reactions can be carried out together, orstepwise. For example, Rebaudioside M can be produced from RebaudiosideD or Rebaudioside E that is commercially available as an enrichedextract or produced via biosynthesis, with the addition ofstoichiometric or excess amounts of UDP-glucose and UGT76G1. As analternative, Rebaudioside D and Rebaudioside M can be produced fromsteviol glycoside extracts that are enriched for stevioside andRebaudioside A, using EUGT11 and a suitable UGT76G1 enzyme. In someembodiments, phosphatases are used to remove secondary products andimprove reaction yields. UGTs and other enzymes for in vitro reactionscan be provided in soluble forms or in immobilized forms.

In some embodiments, Rebaudioside M can be produced using whole cellsthat are fed raw materials that contain precursor molecules such assteviol and/or steviol glycosides, including mixtures of steviolglycosides derived from plant extracts. The raw materials can be fedduring cell growth or after cell growth. The whole cells can be insuspension or immobilized. The whole cells can be entrapped in beads,for example calcium or sodium alginate beads. The whole cells can belinked to a hollow fiber tube reactor system. The whole cells can beconcentrated and entrapped within a membrane reactor system. The wholecells can be in fermentation broth or in a reaction buffer. Similarmethodology can be applied to fermentation of recombinant cells.

In some embodiments, a permeabilizing agent is utilized for efficienttransfer of substrate into the cells. In some embodiments, the cells arepermeabilized with a solvent such as toluene, or with a detergent suchas Triton-X or Tween. In some embodiments, the cells are permeabilizedwith a surfactant, for example a cationic surfactant such ascetyltrimethylammonium bromide (CTAB). In some embodiments, the cellsare permeabilized with periodic mechanical shock such as electroporationor a slight osmotic shock. The cells can contain one recombinant UGT ormultiple recombinant UGTs. For example, the cells can contain UGT76G1,91D2e, 85C2, 74G1 and EUGT11 such that mixtures of steviol and/orsteviol glycosides are efficiently converted to Rebaudioside M. In someembodiments, the whole cells are the host cells described below. In someembodiments, the whole cells are a Gram-negative bacterium such as E.coli. In some embodiments, the whole cell is a Gram-positive bacteriumsuch as Bacillus. In some embodiments, the whole cell is a fungalspecies such as Aspergillus, or yeast such as Saccharomyces. In someembodiments, the term “whole cell biocatalysis” is used to refer to theprocess in which the whole cells are grown as described above (e.g., ina medium and optionally permeabilized) and a substrate such asRebaudioside D, Rebaudioside E, or stevioside is provided and convertedto the end product using the enzymes from the cells. The cells can orcannot be viable, and can or cannot be growing during the bioconversionreactions. In contrast, in fermentation, the cells are cultured in agrowth medium and fed a carbon and energy source such as glucose and theend product is produced with viable cells.

C. Other Polypeptides

Genes for additional polypeptides whose expression facilitates moreefficient or larger scale production of steviol or a steviol glycosidecan also be introduced into a recombinant host. For example, arecombinant microorganism, plant, or plant cell can also contain one ormore genes encoding a geranylgeranyl diphosphate synthase (GGPPS, alsoreferred to as GGDPS). As another example, the recombinant host cancontain one or more genes encoding a rhamnose synthetase, or one or moregenes encoding a UDP-glucose dehydrogenase and/or a UDP-glucuronic aciddecarboxylase. As another example, a recombinant host can also containone or more genes encoding a cytochrome P450 reductase (CPR). Expressionof a recombinant CPR facilitates the cycling of NADP+ to regenerateNADPH, which is utilized as a cofactor for terpenoid biosynthesis. Othermethods can be used to regenerate NADHP levels as well. In circumstanceswhere NADPH becomes limiting, for example, strains can be furthermodified to include exogenous transhydrogenase genes. See, e.g., Saueret al., 2004, J. Biol. Chem. 279: 6613-6619. Other methods are known tothose with skill in the art to reduce or otherwise modify the ratio ofNADH/NADPH such that the desired cofactor level is increased.

As another example the recombinant host can contain one or more genesencoding a sucrose synthase, and additionally can contain sucrose uptakegenes if desired. The sucrose synthase reaction can be used to increasethe UDP-glucose pool in a fermentation host, or in a whole cellbioconversion process. This regenerates UDP-glucose from UDP producedduring glycosylation and sucrose, allowing for efficient glycosylation.In some organisms, disruption of the endogenous invertase isadvantageous to prevent degradation of sucrose. For example, the S.cerevisiae SUC2 invertase can be disrupted. The sucrose synthase (SUS)can be from any suitable organism. For example, a sucrose synthasecoding sequence from, without limitation, A. thaliana, S. rebaudiana, orC. arabica can be cloned into an expression plasmid under control of asuitable promoter, and expressed in a host (e.g., a microorganism or aplant). The sucrose synthase can be expressed in such a strain incombination with a sucrose transporter (e.g., the A. thaliana SUC1transporter or a functional homolog thereof) and one or more UGTs (e.g.,UGT85C2, UGT74G1, UGT76G1, and UGT91D2e, EUGT11 or functional homologsthereof). Culturing the host in a medium that contains sucrose canpromote production of UDP-glucose, as well as one or more glucosides(e.g., steviol glycosides).

Expression of the ERG9 gene, which encodes squalene synthase (SQS), alsocan be reduced in recombinant hosts such that there is a build-up ofprecursors to squalene synthase in the recombinant host. SQS isclassified under EC 2.5.1.21 and is the first committed enzyme of thebiosynthesis pathway that leads to the production of sterols. Itcatalyzes the synthesis of squalene from farnesyl pyrophosphate via theintermediate presqualene pyrophosphate, wherein two units of farnesylpyrophosphate are converted into squalene. This enzyme is a branch pointenzyme in the biosynthesis of terpenoids/isoprenoids and is thought toregulate the flux of isoprene intermediates through the sterol pathway.The enzyme is sometimes referred to as farnesyl-diphosphatefarnesyltransferase (FDFT1). In addition, a recombinant host can havereduced phosphatase activity as discussed herein.

MEP Biosynthesis Polypeptides

As another example, the recombinant host can contain one or more genesencoding one or more enzymes in the MEP pathway or the mevalonatepathway. Such genes are useful because they can increase the flux ofcarbon into the diterpene biosynthesis pathway, producing geranylgeranyldiphosphate from isopentenyl diphosphate and dimethylallyl diphosphategenerated by the pathway. The geranylgeranyl diphosphate so produced canbe directed towards steviol and steviol glycoside biosynthesis due toexpression of steviol biosynthesis polypeptides and steviol glycosidebiosynthesis polypeptides. See, e.g., Brandle et al., 2007,Phytochemistry 68:1855-1863.

In some embodiments, a recombinant host contains one or more genesencoding enzymes involved in the methylerythritol 4-phosphate (MEP)pathway for isoprenoid biosynthesis. Enzymes in the MEP pathway includedeoxyxylulose 5-phosphate synthase (DXS), D-1-deoxyxylulose 5-phosphatereductoisomerase (DXR), 4-diphosphocytidyl-2-C-methyl-D-erythritolsynthase (CMS), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK),4-diphosphocytidyl-2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase(MCS), 1-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate synthase (HDS) and1-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate reductase (HDR). One ormore DXS genes, DXR genes, CMS genes, CMK genes, MCS genes, HDS genesand/or HDR genes can be incorporated into a recombinant microorganism.See, Rodriguez-Concepción and Boronat, Plant Phys. 130: 1079-1089(2002).

Suitable genes encoding DXS, DXR, CMS, CMK, MCS, HDS and/or HDRpolypeptides include those made by E. coli, A. thaliana andSynechococcus leopoliensis. Nucleotide sequences encoding DXRpolypeptides (e.g., SEQ ID NO: 71) are described, for example, in U.S.Pat. No. 7,335,815.

Mevalonate Biosynthesis Polypeptides

S. cerevisiae contains endogenous genes encoding the enzymes of afunctional mevalonate pathway for isoprenoid synthesis. In someembodiments, a recombinant host also contains one or more heterologousgenes encoding enzymes involved in the mevalonate pathway. Genessuitable for transformation into a host encode enzymes in the mevalonatepathway such as a truncated 3-hydroxy-3-methyl-glutaryl (HMG)-CoAreductase (tHMG), and/or a gene encoding a mevalonate kinase (MK),and/or a gene encoding a phosphomevalonate kinase (PMK), and/or a geneencoding a mevalonate pyrophosphate decarboxylase (MPPD). Thus, one ormore HMG-CoA reductase genes, MK genes, PMK genes, and/or MPPD genes canbe incorporated into a recombinant host such as a microorganism.

Suitable genes encoding mevalonate pathway polypeptides are known. Forexample, suitable polypeptides include those made by E. coli, Paracoccusdenitrificans, S. cerevisiae, A. thaliana, Kitasatospora griseola, Homosapiens, Drosophila melanogaster, Gallus gallus, Streptomyces sp.KO-3988, Nicotiana attenuata, Kitasatospora griseola, Heveabrasiliensis, Enterococcus faecium and Haematococcus pluvialis. See,e.g., Table 9, U.S. Pat. Nos. 7,183,089, 5,460,949, and 5,306,862, andPCT Application Nos. PCT/US2012/050021 and PCT/US2011/038967, which areincorporated herein by reference in their entirety.

TABLE 9 Sources of HMG CoA Reductases and other Mevalonate Genes SizeGene Accession# Organism Enzyme (nt) name XM_001467423 LeishmaniaAcetyl-CoA 1323 MEV-4 (amino acid SEQ infantum C-acetyltransferase (ntSEQ ID NO: 72) ID NO: 94) YML075C Saccharomyces Truncated HMG 1584 tHMG1(amino acid SEQ cerevisiae (tHMG1) (nt SEQ ID NO: 73) ID NO: 95)EU263989 Ganoderma 3-HMG-CoA 3681 MEV-11 (amino acid SEQ lucidumreductase (nt SEQ ID NO: 74) ID NO: 96) BC153262 Bos taurus 3-HMG-CoA2667 MEV-12 (amino acid SEQ reductase (nt SEQ ID NO: 75) ID NO: 97)AAD47596 Artemisia 3-HMG-CoA 1704 MEV-13 (amino acid SEQ annua reductase(nt SEQ ID NO: 76) ID NO: 98) AAB62280 Trypanosoma 3-HMG-CoA 1308 MEV-14(amiono acid SEQ cruzi reductase (nt SEQ ID NO: 77) ID NO: 99) CAG41604Staph aureus 3-HMG-CoA 1281 MEV-15 (amino acid SEQ reductase (nt SEQ IDNO: 78) ID NO: 100) DNA2.0 sequence Archaeoglobus 3-HMG-CoA 1311 HMG(amino acid SEQ fulgidus reductase (nt SEQ reductase ID NO: 92) ID NO:101) DNA2.0 sequence Pseudomonas 3-HMG-CoA 1287 HMG (amino acid SEQmevalonii reductase (nt SEQ reductase ID NO: 93) ID NO: 102)

Sucrose Synthase Polypeptides

Sucrose synthase (SUS) can be used as a tool for generating UDP-sugar,in particular UDP-glucose. SUS (EC 2.4.1.13) catalyzes the formation ofUDP-glucose and fructose from sucrose and UDP. UDP generated by thereaction of UGTs thus can be converted by SUS into UDP-glucose in thepresence of sucrose. See, e.g., Chen et al., 2001, J. Am. Chem. Soc.123:8866-8867; Shao et al., 2003, Appl. Env. Microbiol. 69:5238-5242;Masada et al., 2007, FEBS Lett. 581:2562-2566; and Son et al., 2009, J.Microbiol. Biotechnol. 19:709-712.

Sucrose synthases can be used to generate UDP-glucose and remove UDP,facilitating efficient glycosylation of compounds in various systems.For example, yeast deficient in the ability to utilize sucrose can bemade to grow on sucrose by introducing a sucrose transporter and a SUS.For example, S. cerevisiae does not have an efficient sucrose uptakesystem, and relies on extracellular SUC2 to utilize sucrose. Thecombination of disrupting the endogenous S. cerevisiae SUC2 invertaseand expressing recombinant SUS resulted in a yeast strain that was ableto metabolize intracellular but not extracellular sucrose (Riesmeier etal., 1992, EMBO J. 11:4705-4713). The strain was used to isolate sucrosetransporters by transformation with a cDNA expression library andselection of transformants that had gained the ability to take upsucrose.

The combined expression of recombinant sucrose synthase and a sucrosetransporter in vivo can lead to increased UDP-glucose availability andremoval of unwanted UDP. For example, functional expression of arecombinant sucrose synthase, a sucrose transporter, and aglycosyltransferase, in combination with knockout of the natural sucrosedegradation system (SUC2 in the case of S. cerevisiae) can be used togenerate a cell that is capable of producing increased amounts ofglycosylated compounds such as steviol glycosides. This higherglycosylation capability is due to at least (a) a higher capacity forproducing UDP-glucose in a more energy efficient manner, and (b) removalof UDP from growth medium, as UDP can inhibit glycosylation reactions.

The sucrose synthase can be from any suitable organism. For example, asucrose synthase coding sequence from, without limitation, A. thaliana(e.g. SEQ ID NO: 79 or 80), or C. arabica (e.g., SEQ ID NO: 81) (seee.g., SEQ ID NOs: 178, 179, and 180 of PCT/US2012/050021, which isincorporated herein by reference in its entirety) includes the aminoacid sequence of the sucrose transporter SUC1 from A. thaliana (SEQ IDNO: 80), and the amino acid sequence of the sucrose synthase from coffee(SEQ ID NO: 81).

The sucrose synthase can be from any suitable organism. For example, asucrose synthase coding sequence from, without limitation, A. thaliana,S. rebaudiana, or C. arabica (see e.g., SEQ ID NOs: 79-81) can be clonedinto an expression plasmid under control of a suitable promoter, andexpressed in a host (e.g., a microorganism or a plant). A SUS codingsequence can be expressed in a SUC2 (sucrose hydrolyzing enzyme)deficient S. cerevisiae strain, so as to avoid degradation ofextracellular sucrose by the yeast.

The sucrose synthase can be expressed in such a strain in combinationwith a sucrose transporter (e.g., the A. thaliana SUC1 transporter or afunctional homolog thereof) and one or more UGTs (e.g., UGT85C2,UGT74G1, UGT76G1, EUGT11, and UGT91D2e, or functional homologs thereof).Culturing the host in a medium that contains sucrose can promoteproduction of UDP-glucose, as well as one or more glucosides (e.g.,steviol glucoside). It is to be noted that in some cases, a sucrosesynthase and a sucrose transporter can be expressed along with a UGT ina host cell that also is recombinant for production of a particularcompound (e.g., steviol).

Modulating Expression of ERG9 Gene

Expression of the endogenous ERG9 gene can be altered in a recombinanthost described herein using a nucleic acid construct. The construct caninclude two regions that are homologous to parts of the genome sequencewithin the ERG9 promoter or 5′ end of the ERG9 open reading frame (ORF),respectively. The construct can further include a promoter, such aseither the wild type ScKex2 or wild type ScCyc1, and the promoterfurther can include a heterologous insert such as a hairpin at its3′-end. The polypeptide encoded by the ORF advantageously has at least70% identity to a squalene synthase (EC 2.5.1.21) or a biologicallyactive fragment thereof, said fragment having at least 70% sequenceidentity to said squalene synthase in a range of overlap of at least 100amino acids. See, for example, PCT/US2012/050021 (incorporated hereinfor all purposes in its entirety).

The heterologous insert can adapt the secondary structure element of ahairpin with a hairpin loop. The heterologous insert sequence has thegeneral formula (I):

-X1-X2-X3-X4-X5, wherein

X2 comprises at least 4 consecutive nucleotides being complementary to,and forming a hairpin secondary structure element with at least 4consecutive nucleotides of X4, and

X3 is optional and if present comprises nucleotides involved in forminga hairpin loop between X2 and X4, and

X1 and X5 individually and optionally comprise one or more nucleotides,and

X2 and X4 can individually consist of any suitable number ofnucleotides, so long as a consecutive sequence of at least 4 nucleotidesof X2 is complementary to a consecutive sequence of at least 4nucleotides of X4. In some embodiments, X2 and X4 consist of the samenumber of nucleotides.

The heterologous insert is long enough to allow a hairpin to becompleted, but short enough to allow limited translation of an ORF thatis present in-frame and immediately 3′ to the heterologous insert.Typically, the heterologous insert is from 10-50 nucleotides in length,e.g., 10-30 nucleotides, 15-25 nucleotides, 17-22 nucleotides, 18-21nucleotides, 18-20 nucleotides, or 19 nucleotides in length. As providedherein:

X2 can for example consist of in the range of 4 to 25 nucleotides, suchas in the range of 4 to 20, 4 to 15, 6 to 12, 8 to 12, or 9 to 11nucleotides.

X4 can for example consist of in the range of 4 to 25 nucleotides, suchas in the range of 4 to 20, 4 to 15, 6 to 12, 8 to 12, or 9 to 11nucleotides.

In some embodiments, X2 consists of a nucleotide sequence that iscomplementary to the nucleotide sequence of X4, all nucleotides of X2are complementary to the nucleotide sequence of X4.

X3 can be absent, i.e., X3 can consist of zero nucleotides. It is alsopossible that X3 consists of in the range of 1 to 5 nucleotides, such asin the range of 1 to 3 nucleotides.

X1 can be absent, i.e., X1 can consist of zero nucleotides. It is alsopossible that X1 consists of in the range of 1 to 25 nucleotides, suchas in the range of 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3nucleotides.

X5 can be absent, i.e., X5 can consist of zero nucleotides. It is alsopossible that X5 can consist of in the range 1 to 5 nucleotides, such asin the range of 1 to 3 nucleotides.

The heterologous insert can be any suitable sequence fulfilling therequirements defined herein. For example, the heterologous insert cancomprise tgaattcgttaacgaattc (SEQ ID NO: 82), tgaattcgttaacgaactc (SEQID NO: 83), tgaattcgttaacgaagtc (SEQ ID NO: 84), or tgaattcgttaacgaaatt(SEQ ID NO: 85).

Without being bound to a particular mechanism, ERG9 expression can bedecreased by at least partly, sterically hindering binding of theribosome to the RNA thus reducing the translation of squalene synthase.Thus, the translation rate of a functional squalene synthase (EC2.5.1.21) can be reduced, for example. Using such a construct also candecrease turnover of farnesyl-pyrophosphate to squalene and/or enhanceaccumulation of a compound selected from the group consisting offarnesyl-pyrophosphate, isopentenyl-pyrophosphate,dimethylallyl-pyrophosphate, geranyl-pyrophosphate andgeranylgeranyl-pyrophosphate.

In some instances it can be advantageous to include a squalene synthaseinhibitor when culturing recombinant hosts described herein. Chemicalinhibition of squalene synthase, e.g., by lapaquistat, is known in theart. Other squalene synthase inhibitors include Zaragozic acid and RPR107393. Thus, in one embodiment the culturing step of the method(s)defined herein are performed in the presence of a squalene synthaseinhibitor.

In some embodiments, the recombinant hosts described herein contain amutation in the ERG9 open reading frame.

In some embodiments, the recombinant hosts described herein contain anERG9[Delta]::HIS3 deletion/insertion allele.

D. Functional Homologs

Functional homologs of the polypeptides described above are alsosuitable for use in producing steviol or steviol glycosides in arecombinant host. A functional homolog is a polypeptide that hassequence similarity to a reference polypeptide, and that carries out oneor more of the biochemical or physiological function(s) of the referencepolypeptide. A functional homolog and the reference polypeptide can benatural occurring polypeptides, and the sequence similarity can be dueto convergent or divergent evolutionary events. As such, functionalhomologs are sometimes designated in the literature as homologs, ororthologs, or paralogs. Variants of a naturally occurring functionalhomolog, such as polypeptides encoded by mutants of a wild type codingsequence, can themselves be functional homologs. Functional homologs canalso be created via site-directed mutagenesis of the coding sequence fora polypeptide, or by combining domains from the coding sequences fordifferent naturally-occurring polypeptides (“domain swapping”).Techniques for modifying genes encoding functional UGT polypeptidesdescribed herein are known and include, inter alia, directed evolutiontechniques, site-directed mutagenesis techniques and random mutagenesistechniques, and can be useful to increase specific activity of apolypeptide, alter substrate specificity, alter expression levels, altersubcellular location, or modify polypeptide:polypeptide interactions ina desired manner. Such modified polypeptides are considered functionalhomologs. The term “functional homolog” is sometimes applied to thenucleic acid that encodes a functionally homologous polypeptide.

Functional homologs can be identified by analysis of nucleotide andpolypeptide sequence alignments. For example, performing a query on adatabase of nucleotide or polypeptide sequences can identify homologs ofsteviol or steviol glycoside biosynthesis polypeptides. Sequenceanalysis can involve BLAST, Reciprocal BLAST, or PSI-BLAST analysis ofnonredundant databases using a GGPPS, a CDPS, a KS, a KO or a KAH aminoacid sequence as the reference sequence. Amino acid sequence is, in someinstances, deduced from the nucleotide sequence. Those polypeptides inthe database that have greater than 40% sequence identity are candidatesfor further evaluation for suitability as a steviol or steviol glycosidebiosynthesis polypeptide. Amino acid sequence similarity allows forconservative amino acid substitutions, such as substitution of onehydrophobic residue for another or substitution of one polar residue foranother. If desired, manual inspection of such candidates can be carriedout in order to narrow the number of candidates to be further evaluated.Manual inspection can be performed by selecting those candidates thatappear to have domains present in steviol biosynthesis polypeptides,e.g., conserved functional domains.

Conserved regions can be identified by locating a region within theprimary amino acid sequence of a steviol or a steviol glycosidebiosynthesis polypeptide that is a repeated sequence, forms somesecondary structure (e.g., helices and beta sheets), establishespositively or negatively charged domains, or represents a protein motifor domain. See, e.g., the Pfam web site describing consensus sequencesfor a variety of protein motifs and domains on the World Wide Web atsanger.ac.uk/Software/Pfam/and pfam.janelia.org/. The informationincluded at the Pfam database is described in Sonnhammer et al., Nucl.Acids Res., 26:320-322 (1998); Sonnhammer et al., Proteins, 28:405-420(1997); and Bateman et al., Nucl. Acids Res., 27:260-262 (1999).Conserved regions also can be determined by aligning sequences of thesame or related polypeptides from closely related species. Closelyrelated species preferably are from the same family. In someembodiments, alignment of sequences from two different species isadequate.

Typically, polypeptides that exhibit at least about 40% amino acidsequence identity are useful to identify conserved regions. Conservedregions of related polypeptides exhibit at least 45% amino acid sequenceidentity (e.g., at least 50%, at least 60%, at least 70%, at least 80%,or at least 90% amino acid sequence identity). In some embodiments, aconserved region exhibits at least 92%, 94%, 96%, 98%, or 99% amino acidsequence identity.

For example, polypeptides suitable for producing steviol glycosides in arecombinant host include functional homologs of EUGT11 (SEQ ID NO: 16),UGT91D2e (SEQ ID NO: 15), UGT91D2m (SEQ ID NO: 86), UGT85C (SEQ ID NO:26), and UGT76G (SEQ ID NO:2). Such homologs have greater than 90%(e.g., at least 95% or 99%) sequence identity to the amino acid sequenceof EUGT11, UGT91D2e, UGT91D2m, UGT85C, or UGT76G disclosed herein or inPCT Application No. PCT/US2012/050021, which is incorporated herein byreference in its entirety. Variants of EUGT11, UGT91D2, UGT85C, andUGT76G polypeptides typically have 10 or fewer amino acid substitutionswithin the primary amino acid sequence, e.g., 7 or fewer amino acidsubstitutions, 5 or conservative amino acid substitutions, or between 1and 5 substitutions. However, in some embodiments, variants of EUGT11,UGT91D2, UGT85C, and UGT76G polypeptides can have 10 or more amino acidsubstitutions (e.g., 10, 15, 20, 25, 30, 35, 10-20, 10-35, 20-30, or25-35 amino acid substitutions). The substitutions can be conservative,or in some embodiments, non-conservative. Non-limiting examples ofnon-conservative changes in UGT91D2e polypeptides include glycine toarginine and tryptophan to arginine. Non-limiting examples ofnon-conservative substitutions in UGT76G polypeptides include valine toglutamic acid, glycine to glutamic acid, glutamine to alanine, andserine to proline. Non-limiting examples of changes to UGT85Cpolypeptides include histidine to aspartic acid, proline to serine,lysine to threonine, and threonine to arginine.

In some embodiments, a useful UGT91D2 homolog can have amino acidsubstitutions (e.g., conservative amino acid substitutions) in regionsof the polypeptide that are outside of predicted loops, e.g., residues20-26, 39-43, 88-95, 121-124, 142-158, 185-198, and 203-214 arepredicted loops in the N-terminal domain and residues 381-386 arepredicted loops in the C-terminal domain of 91D2e (see SEQ ID NO:15).For example, a useful UGT91D2 homolog can include at least one aminoacid substitution at residues 1-19, 27-38, 44-87, 96-120, 125-141,159-184, 199-202, 215-380, or 387-473. In some embodiments, a UGT91D2homolog can have an amino acid substitution at one or more residuesselected from the group consisting of residues 30, 93, 99, 122, 140,142, 148, 153, 156, 195, 196, 199, 206, 207, 211, 221, 286, 343, 427,and 438. For example, a UGT91D2 functional homolog can have an aminoacid substitution at one or more of residues 206, 207, and 343, such asan arginine at residue 206, a cysteine at residue 207, and an arginineat residue 343. Other functional homologs of UGT91D2 can have one ormore of the following: a tyrosine or phenylalanine at residue 30, aproline or glutamine at residue 93, a serine or valine at residue 99, atyrosine or a phenylalanine at residue 122, a histidine or tyrosine atresidue 140, a serine or cysteine at residue 142, an alanine orthreonine at residue 148, a methionine at residue 152, an alanine atresidue 153, an alanine or serine at residue 156, a glycine at residue162, a leucine or methionine at residue 195, a glutamic acid at residue196, a lysine or glutamic acid at residue 199, a leucine or methionineat residue 211, a leucine at residue 213, a serine or phenylalanine atresidue 221, a valine or isoleucine at residue 253, a valine or alanineat residue 286, a lysine or asparagine at residue 427, an alanine atresidue 438, and either an alanine or threonine at residue 462. Inanother embodiment, a UGT91D2 functional homolog contains a methionineat residue 211 and an alanine at residue 286.

In some embodiments, a useful UGT85C homolog can have one or more aminoacid substitutions at residues 9, 10, 13, 15, 21, 27, 60, 65, 71, 87,91, 220, 243, 270, 289, 298, 334, 336, 350, 368, 389, 394, 397, 418,420, 440, 441, 444, and 471. Non-limiting examples of useful UGT85Chomologs include polypeptides having substitutions (with respect to SEQID NO: 26) at residue 65 (e.g., a serine at residue 65), at residue 65in combination with residue 15 (a leucine at residue 15), 270 (e.g., amethionine, arginine, or alanine at residue 270), 418 (e.g., a valine atresidue 418), 440 (e.g., an aspartic acid at residue at residue 440), or441 (e.g., an asparagine at residue 441); residues 13 (e.g., aphenylalanine at residue 13), 15, 60 (e.g., an aspartic acid at residue60), 270, 289 (e.g., a histidine at residue 289), and 418; substitutionsat residues 13, 60, and 270; substitutions at residues 60 and 87 (e.g.,a phenylalanine at residue 87); substitutions at residues 65, 71 (e.g.,a glutamine at residue 71), 220 (e.g., a threonine at residue 220), 243(e.g., a tryptophan at residue 243), and 270; substitutions at residues65, 71, 220, 243, 270, and 441; substitutions at residues 65, 71, 220,389 (e.g., a valine at residue 389), and 394 (e.g., a valine at residue394); substitutions at residues 65, 71, 270, and 289; substitutions atresidues 220, 243, 270, and 334 (e.g., a serine at residue 334); orsubstitutions at residues 270 and 289. The following amino acidmutations did not result in a loss of activity in 85C2 polypeptides:V13F, F15L, H60D, A65S, E71Q, 187F, K220T, R243W, T270M, T270R, Q289H,L334S, A389V, I394V, P397S, E418V, G440D, and H441N. Additionalmutations that were seen in active clones include K9E, K10R, Q21H, M27V,L91P, Y298C, K350T, H368R, G420R, L431P, R444G, and M471T. In someembodiments, an UGT85C2 contains substitutions at positions 65 (e.g., aserine), 71 (a glutamine), 270 (a methionine), 289 (a histidine), and389 (a valine).

In some embodiments, a useful UGT76G1 homolog (SEQ ID NO: 2) can haveone or more amino acid substitutions at residues 29, 74, 87, 91, 116,123, 125, 126, 130, 145, 192, 193, 194, 196, 198, 199, 200, 203, 204,205, 206, 207, 208, 266, 273, 274, 284, 285, 291, 330, 331, and 346 (seeTABLE 10). Non-limiting examples of useful UGT76G1 homologs includepolypeptides having substitutions at residues 74, 87, 91, 116, 123, 125,126, 130, 145, 192, 193, 194, 196, 198, 199, 200, 203, 204, 205, 206,207, 208, and 291; residues 74, 87, 91, 116, 123, 125, 126, 130, 145,192, 193, 194, 196, 198, 199, 200, 203, 204, 205, 206, 207, 208, 266,273, 274, 284, 285, and 291; or residues 74, 87, 91, 116, 123, 125, 126,130, 145, 192, 193, 194, 196, 198, 199, 200, 203, 204, 205, 206, 207,208, 266, 273, 274, 284, 285, 291, 330, 331, and 346. See, Table 10.

TABLE 10 Clone Variants 76G_G7 M29I, V74E, V87G, L91P, G116E, A123T,Q125A, I126L, T130A, V145M, C192S, S193A, F194Y, M196N, K198Q, K199I,Y200L, Y203I, F204L, E205G, N206K, I207M, T208I, P266Q, S273P, R274S,G284T, T285S, 287-3 bp deletion, L330V, G331A, L346I 76G_H12 M29I, V74E,V87G, L91P, G116E, A123T, Q125A, I126L, T130A, V145M, C192S, S193A,F194Y, M196N, K198Q, K199I, Y200L, Y203I, F204L, E205G, N206K, I207M,T208I, P266Q, S273P, R274S, G284T, T285S, 287-3 bp deletion 76G_C4 M29I,V74E, V87G, L91P, G116E, A123T, Q125A, I126L, T130A, V145M, C192S,S193A, F194Y, M196N, K198Q, K199I, Y200L, Y203I, F204L, E205G, N206K,I207M, T208I

Methods to modify the substrate specificity of, for example, EUGT11 orUGT91D2e, are known to those skilled in the art, and include withoutlimitation site-directed/rational mutagenesis approaches, randomdirected evolution approaches and combinations in which randommutagenesis/saturation techniques are performed near the active site ofthe enzyme. For example see Sarah A. Osmani, et al., Phytochemistry 70(2009) 325-347.

A candidate sequence typically has a length that is from 80 percent to200 percent of the length of the reference sequence, e.g., 82, 85, 87,89, 90, 93, 95, 97, 99, 100, 105, 110, 115, 120, 130, 140, 150, 160,170, 180, 190, or 200 percent of the length of the reference sequence. Afunctional homolog polypeptide typically has a length that is from 95percent to 105 percent of the length of the reference sequence, e.g.,90, 93, 95, 97, 99, 100, 105, 110, 115, or 120 percent of the length ofthe reference sequence, or any range between. A percent identity for anycandidate nucleic acid or polypeptide relative to a reference nucleicacid or polypeptide can be determined as follows. A reference sequence(e.g., a nucleic acid sequence or an amino acid sequence describedherein) is aligned to one or more candidate sequences using the computerprogram ClustalW (version 1.83, default parameters), which allowsalignments of nucleic acid or polypeptide sequences to be carried outacross their entire length (global alignment). Chenna et al., NucleicAcids Res., 31(13):3497-500 (2003).

ClustalW calculates the best match between a reference and one or morecandidate sequences, and aligns them so that identities, similaritiesand differences can be determined. Gaps of one or more residues can beinserted into a reference sequence, a candidate sequence, or both, tomaximize sequence alignments. For fast pairwise alignment of nucleicacid sequences, the following default parameters are used: word size: 2;window size: 4; scoring method: percentage; number of top diagonals: 4;and gap penalty: 5. For multiple alignment of nucleic acid sequences,the following parameters are used: gap opening penalty: 10.0; gapextension penalty: 5.0; and weight transitions: yes. For fast pairwisealignment of protein sequences, the following parameters are used: wordsize: 1; window size: 5; scoring method: percentage; number of topdiagonals: 5; gap penalty: 3. For multiple alignment of proteinsequences, the following parameters are used: weight matrix: blosum; gapopening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps:on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gin, Glu, Arg, andLys; residue-specific gap penalties: on. The ClustalW output is asequence alignment that reflects the relationship between sequences.ClustalW can be run, for example, at the Baylor College of MedicineSearch Launcher site on the World Wide Web(searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at theEuropean Bioinformatics Institute site on the World Wide Web(ebi.ac.uk/clustalw).

To determine percent identity of a candidate nucleic acid or amino acidsequence to a reference sequence, the sequences are aligned usingClustalW, the number of identical matches in the alignment is divided bythe length of the reference sequence, and the result is multiplied by100. It is noted that the percent identity value can be rounded to thenearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are roundeddown to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded upto 78.2.

It will be appreciated that functional UGTs can include additional aminoacids that are not involved in glucosylation or other enzymaticactivities carried out by the enzyme, and thus such a polypeptide can belonger than would otherwise be the case. For example, a EUGT11polypeptide can include a purification tag (e.g., HIS tag or GST tag), achloroplast transit peptide, a mitochondrial transit peptide, anamyloplast peptide, signal peptide, or a secretion tag added to theamino or carboxy terminus. In some embodiments, a EUGT11 polypeptideincludes an amino acid sequence that functions as a reporter, e.g., agreen fluorescent protein or yellow fluorescent protein.

II. Steviol and Steviol Glycoside Biosynthesis Nucleic Acids

A recombinant gene encoding a polypeptide described herein comprises thecoding sequence for that polypeptide, operably linked in senseorientation to one or more regulatory regions suitable for expressingthe polypeptide. Because many microorganisms are capable of expressingmultiple gene products from a polycistronic mRNA, multiple polypeptidescan be expressed under the control of a single regulatory region forthose microorganisms, if desired. A coding sequence and a regulatoryregion are considered to be operably linked when the regulatory regionand coding sequence are positioned so that the regulatory region iseffective for regulating transcription or translation of the sequence.Typically, the translation initiation site of the translational readingframe of the coding sequence is positioned between one and about fiftynucleotides downstream of the regulatory region for a monocistronicgene.

In many cases, the coding sequence for a polypeptide described herein isidentified in a species other than the recombinant host, i.e., is aheterologous nucleic acid. Thus, if the recombinant host is amicroorganism, the coding sequence can be from other prokaryotic oreukaryotic microorganisms, from plants or from animals. In some case,however, the coding sequence is a sequence that is native to the hostand is being reintroduced into that organism. A native sequence canoften be distinguished from the naturally occurring sequence by thepresence of non-natural sequences linked to the exogenous nucleic acid,e.g., non-native regulatory sequences flanking a native sequence in arecombinant nucleic acid construct. In addition, stably transformedexogenous nucleic acids typically are integrated at positions other thanthe position where the native sequence is found.

“Regulatory region” refers to a nucleic acid having nucleotide sequencesthat influence transcription or translation initiation and rate, andstability and/or mobility of a transcription or translation product.Regulatory regions include, without limitation, promoter sequences,enhancer sequences, response elements, protein recognition sites,inducible elements, protein binding sequences, 5′ and 3′ untranslatedregions (UTRs), transcriptional start sites, termination sequences,polyadenylation sequences, introns, and combinations thereof. Aregulatory region typically comprises at least a core (basal) promoter.A regulatory region also can include at least one control element, suchas an enhancer sequence, an upstream element or an upstream activationregion (UAR). A regulatory region is operably linked to a codingsequence by positioning the regulatory region and the coding sequence sothat the regulatory region is effective for regulating transcription ortranslation of the sequence. For example, to operably link a codingsequence and a promoter sequence, the translation initiation site of thetranslational reading frame of the coding sequence is typicallypositioned between one and about fifty nucleotides downstream of thepromoter. A regulatory region can, however, be positioned as much asabout 5,000 nucleotides upstream of the translation initiation site, orabout 2,000 nucleotides upstream of the transcription start site.

The choice of regulatory regions to be included depends upon severalfactors, including, but not limited to, efficiency, selectability,inducibility, desired expression level, and preferential expressionduring certain culture stages. It is a routine matter for one of skillin the art to modulate the expression of a coding sequence byappropriately selecting and positioning regulatory regions relative tothe coding sequence. It will be understood that more than one regulatoryregion can be present, e.g., introns, enhancers, upstream activationregions, transcription terminators, and inducible elements.

One or more genes can be combined in a recombinant nucleic acidconstruct in “modules” useful for a discrete aspect of steviol and/orsteviol glycoside production. Combining a plurality of genes in amodule, particularly a polycistronic module, facilitates the use of themodule in a variety of species. For example, a steviol biosynthesis genecluster, or a UGT gene cluster, can be combined in a polycistronicmodule such that, after insertion of a suitable regulatory region, themodule can be introduced into a wide variety of species. As anotherexample, a UGT gene cluster can be combined such that each UGT codingsequence is operably linked to a separate regulatory region, to form aUGT module. Such a module can be used in those species for whichmonocistronic expression is necessary or desirable. In addition to genesuseful for steviol or steviol glycoside production, a recombinantconstruct typically also contains an origin of replication, and one ormore selectable markers for maintenance of the construct in appropriatespecies.

It will be appreciated that because of the degeneracy of the geneticcode, a number of nucleic acids can encode a particular polypeptide;i.e., for many amino acids, there is more than one nucleotide tripletthat serves as the codon for the amino acid. Thus, codons in the codingsequence for a given polypeptide can be modified such that optimalexpression in a particular host is obtained, using appropriate codonbias tables for that host (e.g., microorganism). As isolated nucleicacids, these modified sequences can exist as purified molecules and canbe incorporated into a vector or a virus for use in constructing modulesfor recombinant nucleic acid constructs.

In some cases, it is desirable to inhibit one or more functions of anendogenous polypeptide in order to divert metabolic intermediatestowards steviol or steviol glycoside biosynthesis. For example, it canbe desirable to downregulate synthesis of sterols in a yeast strain inorder to further increase steviol or steviol glycoside production, e.g.,by downregulating squalene epoxidase. As another example, it can bedesirable to inhibit degradative functions of certain endogenous geneproducts, e.g., glycohydrolases that remove glucose moieties fromsecondary metabolites or phosphatases as discussed herein. As anotherexample, expression of membrane transporters involved in transport ofsteviol glycosides can be inhibited, such that secretion of glycosylatedsteviosides is inhibited. Such regulation can be beneficial in thatsecretion of steviol glycosides can be inhibited for a desired period oftime during culture of the microorganism, thereby increasing the yieldof glycoside product(s) at harvest. In such cases, a nucleic acid thatinhibits expression of the polypeptide or gene product can be includedin a recombinant construct that is transformed into the strain.Alternatively, mutagenesis can be used to generate mutants in genes forwhich it is desired to inhibit function.

III. Hosts Microorganisms

Recombinant hosts can be used to express polypeptides for the productionof steviol glycosides, including mammalian, insect, and plant cells. Anumber of prokaryotes and eukaryotes are also suitable for use inconstructing the recombinant microorganisms described herein, e.g.,gram-negative bacteria, yeast and fungi. A species and strain selectedfor use as a steviol or steviol glycoside production strain is firstanalyzed to determine which production genes are endogenous to thestrain and which genes are not present. Genes for which an endogenouscounterpart is not present in the strain are assembled in one or morerecombinant constructs, which are then transformed into the strain inorder to supply the missing function(s).

Exemplary prokaryotic and eukaryotic species are described in moredetail below. However, it will be appreciated that other species can besuitable. For example, suitable species can be in a genus selected fromthe group consisting of Agaricus, Aspergillus, Bacillus, Candida,Corynebacterium, Eremothecium, Escherichia, Fusarium/Gibberella,Kluyveromyces, Laetiporus, Lentinus, Phaffia, Phanerochaete, Pichia,Physcomitrella, Rhodoturula, Saccharomyces, Schizosaccharomyces,Sphaceloma, Xanthophyllomyces and Yarrowia. Exemplary species from suchgenera include Lentinus tigrinus, Laetiporus sulphureus, Phanerochaetechrysosporium, Pichia pastoris, Cyberlindnera jadinii, Physcomitrellapatens, Rhodoturula glutinis 32, Rhodoturula mucilaginosa, Phaffiarhodozyma U BV-AX, Xanthophyllomyces dendrorhous, Fusariumfujikuroi/Gibberella fujikuroi, Candida utilis, Candida glabrata,Candida albicans, and Yarrowia lipolytica. In some embodiments, amicroorganism can be an Ascomycete such as Gibberella fujikuroi,Kluyveromyces lactis, Schizosaccharomyces pombe, Aspergillus niger,Yarrowia lipolytica, Ashbya gossypii, or Saccharomyces cerevisiae. Insome embodiments, a microorganism can be a prokaryote such asEscherichia coli, Rhodobacter sphaeroides, or Rhodobacter capsulatus. Itwill be appreciated that certain microorganisms can be used to screenand test genes of interest in a high throughput manner, while othermicroorganisms with desired productivity or growth characteristics canbe used for large-scale production of steviol glycosides.

Saccharomyces cerevisiae

Saccharomyces cerevisiae is a widely used chassis organism in syntheticbiology, and can be used as the recombinant microorganism platform.There are libraries of mutants, plasmids, detailed computer models ofmetabolism and other information available for S. cerevisiae, allowingfor rational design of various modules to enhance product yield. Methodsare known for making recombinant microorganisms.

A steviol biosynthesis gene cluster can be expressed in yeast using anyof a number of known promoters. Strains that overproduce terpenes areknown and can be used to increase the amount of geranylgeranyldiphosphate available for steviol and steviol glycoside production.Aspergillus spp.

Aspergillus species such as A. oryzae, A. niger and A. sojae are widelyused microorganisms in food production, and can also be used as therecombinant microorganism platform. Nucleotide sequences are availablefor genomes of A. nidulans, A. fumigatus, A. oryzae, A. clavatus, A.flavus, A. niger, and A. terreus, allowing rational design andmodification of endogenous pathways to enhance flux and increase productyield. Metabolic models have been developed for Aspergillus, as well astranscriptomic studies and proteomics studies. A. niger is cultured forthe industrial production of a number of food ingredients such as citricacid and gluconic acid, and thus species such as A. niger are generallysuitable for the production of food ingredients such as steviol andsteviol glycosides.

Escherichia coli

Escherichia coli, another widely used platform organism in syntheticbiology, can also be used as the recombinant microorganism platform.Similar to Saccharomyces, there are libraries of mutants, plasmids,detailed computer models of metabolism and other information availablefor E. coli, allowing for rational design of various modules to enhanceproduct yield. Methods similar to those described above forSaccharomyces can be used to make recombinant E. coli microorganisms.

Agaricus, Gibberella, and Phanerochaete spp.

Agaricus, Gibberella, and Phanerochaete spp. can be useful because theyare known to produce large amounts of gibberellin in culture. Thus, theterpene precursors for producing large amounts of steviol and steviolglycosides are already produced by endogenous genes. Thus, modulescontaining recombinant genes for steviol or steviol glycosidebiosynthesis polypeptides can be introduced into species from suchgenera without the necessity of introducing mevalonate or MEP pathwaygenes.

Arxula adeninivorans (Blastobotrys adeninivorans)

Arxula adeninivorans is a dimorphic yeast (it grows as a budding yeastlike the baker's yeast up to a temperature of 42° C., above thisthreshold it grows in a filamentous form) with unusual biochemicalcharacteristics. It can grow on a wide range of substrates and canassimilate nitrate. It has successfully been applied to the generationof strains that can produce natural plastics or the development of abiosensor for estrogens in environmental samples.

Yarrowia lipolytica

Yarrowia lipolytica is a dimorphic yeast (see Arxula adeninivorans) thatcan grow on a wide range of substrates. It has a high potential forindustrial applications but there are no recombinant productscommercially available yet.

Rhodobacter spp.

Rhodobacter can be used as the recombinant microorganism platform.Similar to E. coli, there are libraries of mutants available as well assuitable plasmid vectors, allowing for rational design of variousmodules to enhance product yield. Isoprenoid pathways have beenengineered in membraneous bacterial species of Rhodobacter for increasedproduction of carotenoid and CoQ10. See, U.S. Patent Publication Nos.20050003474 and 20040078846. Methods similar to those described abovefor E. coli can be used to make recombinant Rhodobacter microorganisms.

Candida boidinii

Candida boidinii is a methylotrophic yeast (it can grow on methanol).Like other methylotrophic species such as Hansenula polymorpha andPichia pastoris, it provides an excellent platform for the production ofheterologous proteins. Yields in a multigram range of a secreted foreignprotein have been reported. A computational method, IPRO, recentlypredicted mutations that experimentally switched the cofactorspecificity of Candida boidinii xylose reductase from NADPH to NADH.

Hansenula polymorpha (Pichia angusta)

Hansenula polymorpha is another methylotrophic yeast (see Candidaboidinii). It can furthermore grow on a wide range of other substrates;it is thermo-tolerant and can assimilate nitrate (see also Kluyveromyceslactis). It has been applied to the production of hepatitis B vaccines,insulin and interferon alpha-2a for the treatment of hepatitis C,furthermore to a range of technical enzymes.

Kluyveromyces lactis

Kluyveromyces lactis is yeast regularly applied to the production ofkefir. It can grow on several sugars, most importantly on lactose whichis present in milk and whey. It has successfully been applied amongothers to the production of chymosin (an enzyme that is usually presentin the stomach of calves) for the production of cheese. Production takesplace in fermenters on a 40,000 L scale.

Pichia pastoris

Pichia pastoris is a methylotrophic yeast (see Candida boidinii andHansenula polymorpha). It provides an efficient platform for theproduction of foreign proteins. Platform elements are available as a kitand it is worldwide used in academia for the production of proteins.Strains have been engineered that can produce complex human N-glycan(yeast glycans are similar but not identical to those found in humans).

Physcomitrella spp.

Physcomitrella mosses, when grown in suspension culture, havecharacteristics similar to yeast or other fungal cultures. This generais becoming an important type of cell for production of plant secondarymetabolites, which can be difficult to produce in other types of cells.

IV. Methods of Producing Steviol Glycosides

Recombinant hosts described herein can be used in methods to producesteviol glycosides such as Rebaudioside M. For example, if therecombinant host is a microorganism, the method can include growing therecombinant microorganism in a culture medium under conditions in whichsteviol and/or steviol glycoside biosynthesis genes are expressed. Therecombinant microorganism can be grown in a fed batch or continuousprocess. Typically, the recombinant microorganism is grown in afermentor at a defined temperature(s) for a desired period of time. Incertain embodiments, microorganisms include, but are not limited to S.cerevisiae, A. niger, A. oryzae, E. coli, L. lactis and B. subtilis. Theconstructed and genetically engineered microorganisms provided by theinvention can be cultivated using conventional fermentation processes,including, inter alia, chemostat, batch, fed-batch cultivations,continuous perfusion fermentation, and continuous perfusion cellculture.

Depending on the particular microorganism used in the method, otherrecombinant genes such as isopentenyl biosynthesis genes and terpenesynthase and cyclase genes can also be present and expressed. Levels ofsubstrates and intermediates, e.g., isopentenyl diphosphate,dimethylallyl diphosphate, geranylgeranyl diphosphate, kaurene andkaurenoic acid, can be determined by extracting samples from culturemedia for analysis according to published methods.

After the recombinant microorganism has been grown in culture for thedesired period of time, steviol and/or one or more steviol glycosidescan then be recovered from the culture using various techniques known inthe art. In some embodiments, a permeabilizing agent can be added to aidthe feedstock entering into the host and product getting out. If therecombinant host is a plant or plant cells, steviol or steviolglycosides can be extracted from the plant tissue using varioustechniques known in the art. For example, a crude lysate of the culturedmicroorganism or plant tissue can be centrifuged to obtain asupernatant. The resulting supernatant can then be applied to achromatography column, e.g., a C18 column such as Aqua® C18 column fromPhenomenex or a Synergi™ Hydro RP 80 Å column, and washed with water toremove hydrophilic compounds, followed by elution of the compound(s) ofinterest with a solvent such as acetonitrile or methanol. Thecompound(s) can then be further purified by preparative HPLC. See alsoWO 2009/140394.

The amount of steviol glycoside (e.g., Rebaudioside M) produced can befrom about 1 mg/L to about 2800 mg/L, e.g., about 1 to about 10 mg/L,about 3 to about 10 mg/L, about 5 to about 20 mg/L, about 10 to about 50mg/L, about 10 to about 100 mg/L, about 25 to about 500 mg/L, about 100to about 1,500 mg/L, or about 200 to about 1,000 mg/L. In general,longer culture times will lead to greater amounts of product. Thus, therecombinant microorganism can be cultured for from 1 day to 7 days, from1 day to 5 days, from 3 days to 5 days, about 3 days, about 4 days, orabout 5 days.

It will be appreciated that the various genes and modules discussedherein can be present in two or more recombinant microorganisms ratherthan a single microorganism. When a plurality of recombinantmicroorganisms is used, they can be grown in a mixed culture to producesteviol and/or steviol glycosides. For example, a first microorganismcan comprise one or more biosynthesis genes for producing steviol whilea second microorganism comprises steviol glycoside biosynthesis genes.Alternatively, the two or more microorganisms each can be grown in aseparate culture medium and the product of the first culture medium,e.g., steviol, can be introduced into second culture medium to beconverted into a subsequent intermediate, or into an end product such asRebaudioside A. The product produced by the second, or finalmicroorganism is then recovered. It will also be appreciated that insome embodiments, a recombinant microorganism is grown using nutrientsources other than a culture medium and utilizing a system other than afermentor.

Steviol glycosides do not necessarily have equivalent performance indifferent food systems. It is therefore desirable to have the ability todirect the synthesis to steviol glycoside compositions of choice.Recombinant hosts described herein can produce compositions that areselectively enriched for specific steviol glycosides (e.g., RebaudiosideM) and have a consistent taste profile. Thus, the recombinantmicroorganisms, plants, and plant cells described herein can facilitatethe production of compositions that are tailored to meet the sweeteningprofile desired for a given food product and that have a proportion ofeach steviol glycoside that is consistent from batch to batch.Microorganisms described herein do not produce the undesired plantbyproducts found in Stevia extracts. Thus, steviol glycosidecompositions produced by the recombinant microorganisms described hereinare distinguishable from compositions derived from Stevia plants.

V. Food Products

The steviol glycosides obtained by the methods disclosed herein can beused to make food and beverage products, dietary supplements andsweetener compositions. For example, substantially pure steviolglycoside such as Rebaudioside M can be included in food products suchas ice cream, carbonated beverages, fruit juices, yogurts, baked goods,chewing gums, hard and soft candies, and sauces. Substantially puresteviol glycoside also can be included in non-food products such aspharmaceutical products, medicinal products, dietary supplements andnutritional supplements. Substantially pure steviol glycosides can alsobe included in animal feed products for both the agriculture industryand the companion animal industry. Alternatively, a mixture of steviolglycosides can be made by culturing recombinant microorganismsseparately or growing different plants/plant cells, each producing aspecific steviol or steviol glycoside, recovering the steviol or steviolglycoside in substantially pure form from each microorganism orplant/plant cells and then combining the compounds to obtain a mixturecontaining each compound in the desired proportion (e.g., Rebaudioside Mwith one or more other steviol glycosides). The recombinantmicroorganisms, plants, and plant cells described herein permit moreprecise and consistent mixtures to be obtained compared to currentStevia products. In another alternative, a substantially pure steviolglycoside can be incorporated into a food product along with othersweeteners, e.g. saccharin, dextrose, sucrose, fructose, erythritol,aspartame, sucralose, monatin, or acesulfame potassium. The weight ratioof steviol glycoside relative to other sweeteners can be varied asdesired to achieve a satisfactory taste in the final food product. See,e.g., U.S. Patent Publication No. 2007/0128311. In some embodiments, thesteviol glycoside can be provided with a flavor (e.g., citrus) as aflavor modulator.

Compositions produced by a recombinant microorganism, plant, or plantcell described herein can be incorporated into food products. Forexample, a steviol glycoside composition produced by a recombinantmicroorganism, plant, or plant cell can be incorporated into a foodproduct in an amount ranging from about 20 mg steviol glycoside/kg foodproduct to about 1800 mg steviol glycoside/kg food product on a dryweight basis, depending on the type of steviol glycoside and foodproduct. For example, a steviol glycoside composition produced by arecombinant microorganism, plant, or plant cell can be incorporated intoa dessert, cold confectionary (e.g., ice cream), dairy product (e.g.,yogurt), or beverage (e.g., a carbonated beverage) such that the foodproduct has a maximum of 500 mg steviol glycoside/kg food on a dryweight basis. A steviol glycoside composition produced by a recombinantmicroorganism, plant, or plant cell can be incorporated into a bakedgood (e.g., a biscuit) such that the food product has a maximum of 300mg steviol glycoside/kg food on a dry weight basis. A steviol glycosidecomposition produced by a recombinant microorganism, plant, or plantcell can be incorporated into a sauce (e.g., chocolate syrup) orvegetable product (e.g., pickles) such that the food product has amaximum of 1000 mg steviol glycoside/kg food on a dry weight basis. Asteviol glycoside composition produced by a recombinant microorganism,plant, or plant cell can be incorporated into bread such that the foodproduct has a maximum of 160 mg steviol glycoside/kg food on a dryweight basis. A steviol glycoside composition produced by a recombinantmicroorganism, plant, or plant cell can be incorporated into a hard orsoft candy such that the food product has a maximum of 1600 mg steviolglycoside/kg food on a dry weight basis. A steviol glycoside compositionproduced by a recombinant microorganism, plant, or plant cell can beincorporated into a processed fruit product (e.g., fruit juices, fruitfilling, jams, and jellies) such that the food product has a maximum of1000 mg steviol glycoside/kg food on a dry weight basis.

In some embodiments, a substantially pure steviol or steviol glycosideis incorporated into a tabletop sweetener or “cup-for-cup” product. Suchproducts typically are diluted to the appropriate sweetness level withone or more bulking agents, e.g., maltodextrins, known to those skilledin the art. Steviol glycoside compositions enriched for Rebaudioside Mcan be package in a sachet, for example, at from 10,000 to 30,000 mgsteviol glycoside/kg product on a dry weight basis, for tabletop use.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES

The Examples that follow are illustrative of specific embodiments of theinvention and various uses thereof. They are set forth for explanatorypurposes only and are not to be taken as limiting the invention.

Example 1: Strain Engineering and Fermentation of EFSC 3044

Yeast strain EFSC 3044 was derived from a wild type Saccharomycescerevisiae strain containing three auxotrophic modifications, namely thedeletions of URA3, LEU2 and HIS3. The strain can be manipulated usingstandard genetic methods and can be used as a regular diploid or haploidyeast strain. The strain was converted to steviol glycosides-producingyeast by genomic-integration of five DNA constructs. Each constructcontained multiple genes and was introduced into the yeast genome byhomologous recombination. Furthermore, the first, second, and fifthconstruct were assembled by homologous recombination in yeast.

The first construct contained eight genes and was inserted in the DPP1locus and disrupted and partially deleted DPP1 (phosphatase). The DNAinserted contained: the Ashbya gossypii TEF1 promoter expressing thenatMX gene (selectable marker) followed by the TEF1 terminator from A.gossypii; Gene Art codon optimized Stevia rebaudiana UGT85C2 (GenBankAAR06916.1; SEQ ID NO:3) expressed from the native yeast GPD1 promoterand followed by the native yeast CYC1 terminator; S. rebaudiana CPR-8(SEQ ID NO:5) expressed using the native yeast TPI1 promoter followed bythe native yeast TDH1 terminator; Arabidopsis thaliana kaurene synthase(SEQ ID NO:6, similar to GenBank AEE36246.1) expressed from the nativeyeast PDC1 promoter and followed by the native yeast FBA1 terminator;synthetic Synechococcus sp. GGPPS (SEQ ID NO: 22, GenBank ABC98596.1)expressed using the native yeast TEF2 promoter and followed by thenative yeast PGI1 terminator; DNA2.0 codon-optimized S. rebaudiana KAHe1(SEQ ID NO:8) expressed from the native yeast TEF1 promoter and followedby the native yeast ENO2 terminator; synthetic S. rebaudiana KO-1 (SEQID NO: 23, GenBank ABA42921.1) expressed using the native yeast FBA1promoter and followed by the native yeast TDH2 terminator; and Zea maystruncated CDPS (SEQ ID NO:133) expressed using the native yeast PGK1promoter and followed by the native yeast ADH2 terminator.

The second construct was inserted at the YPRCA15 locus and contained:the TEF1 promoter from A. gossypii in front of the kanMX gene(selectable marker) followed by the TEF1 terminator from A. gossypii;the Gene Art codon optimized A. thaliana ATR2 (SEQ ID NO: 10) expressedfrom the native yeast PGK1 promoter followed by the native yeast ADH2terminator; S. rebaudiana UGT74G1 (SEQ ID NO:135, GenBank AAR06920.1)expressed from the native yeast TPI1 promoter followed by the nativeyeast TDH1 terminator; Gene Art codon-optimized S. rebaudiana UGT76G1(SEQ ID NO:14, encodes GenBank AAR06912) expressed from the native yeastTEF1 promoter followed by the native yeast ENO2 terminator; and GeneArtcodon-optimized sequence encoding a S. rebaudiana UGT91D2e-b with theamino acid modifications L211M and V286A (SEQ ID NO:15 for UGT91D2eamino acid sequence for the wild type sequence; codon optimizednucleotide sequence is set forth in SEQ ID NO:90) and expressed from thenative yeast GPD1 promoter and followed by the native yeast CYC1terminator. UGT91D2e-b is disclosed herein as SEQ ID NO: 66 withmutations at methionine residue at residue 211 and an alanine residue atresidue 286.

The first and the second construct were combined in the same spore cloneby mating and dissection. This yeast strain was subsequently transformedwith construct three and four in two successive events.

Construct three was integrated between genes PRP5 and YBR238C andcontained the Kluyveromyces lactis leu2 promoter expressing the K.lactis leu2 gene followed by the leu2 terminator from K. lactis, thenative yeast GPD1 promoter expressing the DNA2.0-optimized S. rebaudianaKAHe1 (SEQ ID NO:8) followed by the native yeast CYC1 terminator, andthe native yeast TPI1 promoter expressing the Zea mays truncated CDPS(SEQ ID NO: 133) followed by the native yeast TPI1 terminator.

Construct four was integrated in the genome between genes ECM3 andYOR093C with an expression cassette containing the TEF1 promoter from A.gossypii expressing the K. pneumoniae hphMX gene followed by the TEF1terminator from A. gossypii, Synechococcus sp. GGPPS (SEQ ID NO: 22)expressed from the native yeast GPD1 promoter followed by the nativeyeast CYC1 terminator, and the native yeast TPI1 promoter expressing theA. thaliana KS (SEQ ID NO: 6) followed by the native yeast TPI1terminator.

The four introduced selectable markers natMX, kanMX, K. lactis LEU2 andK. pneumoniae hphMX and the promoters preceding and terminatorssucceeding the selectable marker genes were then removed byrecombination.

In this yeast strain, the fifth construct was inserted and assembled byyeast transformation and homologue recombination. The fifth constructcontained seven genes and was inserted at the YORWA22 locus. The DNAinserted contained: the A. gossypii TEF1 promoter expressing theSchizosaccharomyces Pombe HIS5 gene (selectable marker) followed by theTEF1 terminator from A. gossypii; S. rebaudiana KO-1 (SEQ ID NO: 23,GenBank ABA42921.1) expressed from the native yeast GPD1 promoter andfollowed by the native yeast CYC1 terminator; S. rebaudiana CPR-8 (SEQID NO: 5) expressed using the native yeast TPI1 promoter followed by thenative yeast TDH1 terminator; Arabidopsis thaliana kaurene synthase (SEQID NO: 6, similar to GenBank AEE36246.1) expressed from the native yeastPDC1 promoter and followed by the native yeast FBA1 terminator; a codonoptimized version of the rice gene Os03g0702000 (SEQ ID NO:18, encodingEUGT11) expressed using the native yeast TEF2 promoter and followed bythe native yeast PGI1 terminator; DNA2.0 codon-optimized S. rebaudianaKAHe1 (SEQ ID NO: 8) expressed from the native yeast TEF1 promoter andfollowed by the native yeast ENO2 terminator; and Zea mays truncatedCDPS (SEQ ID NO:133) expressed using the native yeast PGK1 promoter andfollowed by the native yeast ADH2 terminator.

The described yeast strain was made prototrophic by introduction of thetwo plasmids, EPSC2182 and EPSC2308. EPSC2182 was derived from a p415TEFCEN/ARS shuttle plasmid with a LEU2 marker and contains another copy ofS. rebaudiana KAHe1 expressed from the native yeast TEF1 promoter andsucceeded by the native yeast CYC1 terminator. EPSC2308 was ap416TEF-based CEN/ARS shuttle plasmid with the URA3 marker wherein theEUGT11 gene was cloned and expressed from the native yeast TEF1 promoterand succeeded by the native yeast CYC1 terminator. This yeast strain wasthen designated EFSC 3044.

TABLE 11 List of Recombinant Genes in Strain EFSC 3044. Yeast ConstructGene Designation Location No. UGT85C2 Genomic 1 S. rebaudiana CPR-8Genomic 1 A thaliana Kaurene synthase Genomic 1 Synechococcus sp. GGPPSGenomic 1 S. rebaudiana KAHe1 Genomic 1 S. rebaudiana KO-1 Genomic 1 Zeamays truncated CDPS Genomic 1 A. thaliana ATR2 Genomic 2 S. rebaudianaUGT74G1 Genomic 2 S. rebaudiana UGT76G1 Genomic 2 Stevia UGT91D2e-baltered Genomic 2 S. rebaudiana KAHe1 Genomic 3 Zea mays truncated CDPSGenomic 3 Synechococcus sp. GGPPS Genomic 4 A. thaliana Kaurene synthaseGenomic 4 S. rebaudiana KO-1 Genomic 5 S. rebaudiana CPR-8 Genomic 5 Athaliana Kaurene synthase Genomic 5 Os03g0702000 (EUGT11) Genomic 5 S.rebaudiana KAHe1 Genomic 5 Zea mays truncated CDPS Genomic 5 S.rebaudiana KAHe1 Plasmid 6 EUGT11 Plasmid 7

Fed-batch fermentation was carried out aerobically in 2 L (workingvolume) fermenters which included a ˜16 hour growth phase in the basemedium (Synthetic Complete media) followed by ˜100 hours of feeding withglucose utilized as the carbon and energy source combined with tracemetals, vitamins, salts, and Yeast Nitrogen Base (YNB) and/or amino acidsupplementation. The pH was kept near pH 5, and the temperature setpointwas 30° C. The feed rate was controlled to prevent oxygen depletion andto minimize ethanol formation (glucose-limited conditions). Wholeculture samples (without cell removal) were taken and boiled in an equalvolume of DMSO for total glycosides levels.

The following methodology was used to analyze steviol glycosides andsteviol pathway intermediates, unless otherwise indicated. LC-MSanalyses were performed using an UltiMate® 3000 UPLC system (Dionex,Sunnyvale, Calif.) fitted with an Acquity UPLC® BEH C18 column (100×2.1mm, 1.7 μm particles; Waters, Milford, Mass.) connected to a TSQ QuantumAccess (ThermoFisher Scientific) triple quadropole mass spectrometerwith a heated electrospray ion (HESI) source. Elution was carried outusing a mobile phase of eluent B (MeCN with 0.1% Formic acid) and eluentA (water with 0.1% Formic acid) by increasing the gradient from 29->48%B from min 0.0 to 4.0, increasing 48->100% B in min 4.0 to 4.2, holding100% B from min 4.2 to 6.2, and re-equilibrating with 29% eluent B. Theflow rate was 0.4 ml/min and the column temperature was kept at 55° C.Steviol glycosides were detected using SIM (Single Ion Monitoring) inpositive mode with the following m/z-traces in Table 12.

TABLE 12 Summary of Analytical Compounds Detected by LC/MS. Exact m/zCompound Description Mass trace (typical t_(R) in min) Steviol + [M +H]⁺ 481.2 ± 0.5 19-SMG (4.15), 13-SMG 1 Glucose 481.2796 (4.38) [M +Na]⁺ 503.1 ± 0.5 503.2615 Steviol + [M + Na]⁺  665 ± 0.5 Rubusoside(3.04) 2 Glucose 665.3149 Steviol-1,2-bioside (3.48) Steviol-1,3-bioside(4.05) Steviol + [M + Na]⁺ 827.4 ± 0.5 1,2-Stevioside (2.28) 3 Glucose827.3677 1,3-Stevioside (2.82) Rebaudioside B (3.9) Steviol + [M + Na]⁺989.4 ± 0.5 Rebaudioside A (2.23) 4 Glucose 989.4200 Steviol + [M + Na]⁺1151.4 ± 0.5  Rebaudioside D (1.19) 5 Glucose 1151.4728 Steviol + [M +Na]⁺ 1313.5 ± 0.5  Rebaudioside M (1.31) 6 Glucose 1313.5257

The level of steviol glycosides were quantified by comparing withcalibration curves obtained with authentic standards from LGC Standards.For example, standard solutions of 0.5 to 100 μM Rebaudioside A (RebA)were typically utilized to construct a calibration curve. FIG. 5contains representative mass spectra of fermentations that resulted inthe formation of a hexaglycosylated steviol glycoside (retention time1.31, mass traces corresponding to a hexa-glucose steviol glycoside andstevioside).

A modified LC-MS methodology (using a BEH RPshield C18 HPLC column(50×2.1 mm, 1.7 μm particles; Waters, Milford, Mass.) was used toanalyze compounds described in Example 5 and in vitro experiment todetermine relative rates for UGT76G1. The elution was carried out usinga mobile phase of eluent B (MeCN with 0.1% formic acid) and eluent A(water with 0.1% formic acid) by increasing the gradient from 25->47% Bfrom min 0.0 to 4.0, increasing 47->100% B in min 4.0 to 5.0, holding100% B from min 5.0 to 6.5, and finally re-equilibrating with 25% B. Theflow rate was 0.4 ml/min and the column temperature was kept at 35° C. Amodified LC-MS methodology resulted in shorter retention time for thecompounds shown in Table 12. Typical retention times using the modifiedLC-MS methodology (t_(R) in min) were: 3.34 for 19-SMG; 3.54 for 13-SMG;2.55 for Rubusoside; 2.95 for Steviol-1,2-bioside; 3.31 forSteviol-1,3-bioside; 2.04 for 1,2-Stevioside; 2.42 for 1,3-Stevioside;2.91 for Rebaudioside B; 2.03 for Rebaudioside A; 1.1 for RebaudiosideD; and 1.32 for Rebaudioside M.

Example 2: In Vitro Characterization of Reactions that Produce aHexa-Glycosylated Steviol Glycoside

As described in Example 1, a hexa-glucosyl steviol glycoside wasobserved when EUGT11 was expressed at high levels in steviol-glycosideproducing yeast strains. To characterize the reactions that wereoccurring to produce this molecule, further in vitro work was done withindividual UGTs.

UGT76G1 (SEQ ID NO: 1) was cloned into the pET30a plasmid (EMDMillipore). The resulting vector was transformed into an appropriate DE3E. coli strain and transformants were grown and induced according tomanufacturer's protocols. The corresponding fusion protein(6×HIS-tagged) was purified by immobilized metal affinity chromatographyusing standard methods.

Approximately 0.08 μg of purified UGT76G1 per μL of reaction wasincubated with 100 μM RebD, 300 μM UDP-glucose, and 10 U/mL AlkalinePhosphatase (Fermentas/Thermo Fisher, Waltham, Mass.). The reactionswere performed at 30° C. in 20 mM Hepes-NaOH, pH 7.6, for 24 hours.Prior to LC-MS analysis, one volume of 100% DMSO was added to eachreaction and vortexed, and samples were centrifuged at 16,000×g for 1minute.

A new peak appeared during the reaction at a mass corresponding tosteviol+6 glucose moieties, eluting at 1.31 min and corresponding to atrace of one of the hexaglucosyl steviol glycosides found upon theoverexpression of EUGT11 in vivo. This result suggested that UGT76G1 canfurther glycosylate RebD, resulting in a hexaglycoside. It washypothesized that UGT76G1, in addition to making a 1,3-glucose linkagewith the primary glucose at C13 of the steviol backbone, has a secondaryactivity of adding a 1,3-bound glucose to the primary glucose at C19. Itis likely that the only glycosylation site available in RebD availablefor UGT76G1 is the glucose at C19, which would result in the productionof a hexaglycoside designated RebM. The hexaglycoside detected wasisolated and determined to be Rebaudioside M, as shown in Examples 3 and4.

Example 3: Isolation of the Hexa-Glycosylated Molecule

The hexa-glucosyl steviol glycoside product was isolated from afermentation similar to that described in Example 1 for structuralanalysis following the scheme outlined in FIG. 6.

After the fermentation, the culture broth was centrifuged for 30 min at7000 rpm at 4° C. and the supernatant was purified as follows: A glasscolumn was filled with 150 mL HP20 Diaion® resin (Supelco), and analiquot of 300 mL supernatant was loaded on to the column and washedwith 2×250 mL MilliQ water. The glycoside product was eluted by stepwiseincremental increases in the methanol concentration in MilliQ water (in250 mL portions—starting with 0%→10%→40%→60%→80%→100% MeOH). The levelsof steviol glycosides in each fraction were analyzed by LC-MS. The mostpromising fractions (60-80% MeOH) were combined and reduced to total of10 mL using a vacuum evaporator. A glass column filled with 600 mLspherical C18 bonded flash silica gel (45-70 um, 70 Å/Supelco) wasequilibrated with 5% aqueous acetonitrile (Acetronitrile: HPLCgrade—Water: MilliQ). The concentrated residue from the HP20purification was loaded on the column and eluted by stepwise increasesin the acetonitrile contribution. The starting eluent was 5%acetonitrile in water. The level of acetonitrile was raised by 5% perstep (each 400 mL). After reaching 50% acetonitrile 10% steps were made.All fractions were analyzed by LC-MS, pooled according to their steviolglycoside composition, and dried under vacuum. Table 13 contains asummary of the glycosides found in each of the fractions. FIG. 7contains a chromatogram and mass spectra from LC-QTOF analysis of thesemi-purified hexa-glycosylated compound after flash chromatography.

TABLE 13 Summary of Fractionation of Steviol Glycosides. mg FractionDescription 321.1  2-11 RebD and some 6X glycosylated steviol glycoside138.3 12-20 RebD and some 6X glycosylated steviol glycoside 357.4 21-27Bulk of 6X glycosylated steviol glycoside 98.9 28-30 RebA 68.4 31-36Rubusoside, steviol-1,2-bioside 14.8 34-45 Mostly 13-SMG 852.8 WashAcetonitrile wash

Example 4: NMR Confirmation of Structure

To produce a pure sample for NMR, approximately 50 mg of thehexa-glycosylated enriched residue obtained in Example 3 were furtherpurified on a semi-preparative HPLC system. The system was equipped withan Aqua® C18 column (Phenomenex: Dimension 250×21.2 mm, 5 micron).Elution was carried out using a mobile phase of eluent B (MeCN with 0.1%trifluoroacetic acid) and eluent A (water with 0.1% triflouroacidicacid) by increasing the gradient from 1%→50% B from min 0.0 to 21,50→100% min 21.0 to 27.0 and finally washing with 100% B andre-equilibration. The flow rate was 14 mL/min at room temperature. Thefractions were collected by time and analyzed by LC-QTOF-MS for thepresence of steviol glycosides. The system used was a UPLC (Waters)coupled to a MicrOTOFII Mass Spectrometer (Bruker). The column used wasAcquity UPLC® BEH C18, 100×2.1 mm, 1.7 μm (Waters). Mobile phases wereA: 0.1% Formic Acid in water and B: 0.1% Formic Acid in Acetonitrile.The gradient applied was from 1% B to 50% B in 12 minutes and then to100% B in 3 minutes. The flow rate was 0.4 ml/min.

Fraction 93 was utilized for NMR analysis. All NMR experiments wereperformed in DMSO-d6 at 25C using a Bruker Avance III 600 MHz NMRspectrometer equipped with a 1.7 mm cryogenic TCI probe.

The structures were solved by means of standard homo- and heteronuclearmultipulse NMR experiments, namely ¹H, ¹H-COSY, ¹H, ¹³C-HSQC and ¹H,¹³C-HMBC experiments. The NMR data obtained was as follows:

1H NMR (600 MHz, DMSO-d6) δ ppm 0.78 (br. s., 1H) 0.83 (s, 3H) 0.92 (d,J=7.39 Hz, 2H) 0.97-1.04 (m, 1H) 1.17 (s, 3H) 1.30-1.54 (m, 6H) 1.67 (d,J=10.40 Hz, 1H) 1.72-1.86 (m, 4H) 1.92 (d, J=6.49 Hz, 1H) 1.96-2.05 (m,2H) 2.08 (d, J=10.82 Hz, 1H) 2.32 (d, J=12.71 Hz, 1H) 2.91 (t, J=8.82Hz, 1H) 2.95-3.01 (m, 1H) 3.02-3.27 (m, 14H) 3.31-3.55 (m, 10H)3.57-3.86 (m, 10H) 4.47 (d, J=7.86 Hz, 1H) 4.51 (d, J=8.00 Hz, 1H) 4.53(d, J=7.62 Hz, 1H) 4.66 (d, J=7.81 Hz, 1H) 4.73 (br. s., 1H) 4.80 (d,J=7.86 Hz, 1H) 5.11 (br. s., 1H) 5.53 (d, J=8.19 Hz, 1H)

13C NMR (150.91 MHz, DMSO-d6) δ ppm 16.4, 19.4, 19.9, 21.8, 28.3, 36.7,37.2, 39.3, 40.3, 41.5, 41.6, 43.2, 43.7, 47.0, 47.2, 53.2, 57.0, 60.7,61.2, 61.4, 61.8, 62.0, 62.1, 68.5, 68.9, 70.4 (3C), 71.2, 71.6,74.1-74.3 (4C), 74.8, 75.8, 76.9-77.1 (6C), 77.6, 79.6, 85.8, 86.8,87.1, 92.1, 96.2, 102.1, 102.8, 103.2, 103.3, 104.5, 153.1, 175.1

The confirmed structure of RebM (systematic name:13-[P3-D-glucopyranosyl-(1→3)-[β-D-glucopyranosyl-(1→2)]-□β-D-glucopyranosyl-1-oxy]kaur-16-en-18-oic acid,18-[β-D-glucopyranosyl-(1→3)-[β-D-glucopyranosyl-(1→2))]-□β-D-glucopyranosyl-1-ester])is shown in FIG. 2.

Example 5: Isolation and Determination of Additional FermentationProducts of EFSC 3044

In addition to RebM, fermentation of EFSC 3044 resulted in formation ofa di-glycosylated steviol glycoside (13-hydroxy kaur-16-en-18-oic acid,[2-O-β-D-glucopyranosyl-p3-D-glucopyranosyl] ester) with a retentiontime of 2.31 (FIG. 8B) and a tri-glycosylated steviol glycoside(13-hydroxy kaur-16-en-18-oic acid; [2-O-β-D-glucopyranosyl-3-O-:□β-D-glucopyranosyl-β-D-glucopyranosyl] ester) with a retention time of2.15 (FIG. 8C).

These compounds were isolated according to the following method. Afterthe fermentation, the culture broth was centrifuged for 10 min at 5000rpm at 4° C. and the supernatant was purified as follows: A glass columnwas filled with 300 mL HP20 Diaion® resin (Supelco), and an aliquot of1700 mL supernatant was loaded on to the column and washed with 3.5Litres of ddH2O. The compounds were eluted by using 2 L MeOH andfractions of 500 mL each collected. After LC-MS analysis, the fractionscontaining the majority of the target compounds were pooled andevaporated on a rotary evaporation system (Rotavap, Buchi, Switzerland)yielding 1.85 grams of dark grey material. The crude extract wasre-dissolved in 3.5 mL of DMSO and injected in aliquots of 0.7 mL in asemi-preparative LC-MS for further purification. The column used was aXBridge C18, 19×250 mm, 5 um (Waters Corporation). Mobile phases were A:0.1% TFA in water and B: 0.1% TFA in Acetonitrile. Elution was done by alinear gradient from 1% B to 60% B in 44 min. Fractions of 2.1 mL werecontinuously collected during the run. Fractions collected were analysedby LC-MS in order to evaluate the presence and purity of targetanalytes. Fractions containing compounds identified as ‘Peak 8’ and‘Peak 9’ were neutralized by adding 0.8 mL of NH3 (aq.), pooled anddried by Genevac centrifugal evaporation system.

Structures of these compounds were determined by NMR with the method ofExample 4.

The NMR data obtained for the di-glycosylated steviol glycoside are asfollows:

1H NMR (600 MHz, DMSO-d₆) δ ppm 0.72-0.79 (m, 1H) 0.81 (s, 3H) 0.93 (d,J=8.07 Hz, 1H) 0.98-1.05 (m, 1H) 1.17 (s, 3H) 1.20 (d, J=11.37 Hz, 1H)1.36 (d, J=4.03 Hz, 2H) 1.40-1.52 (m, 1H) 1.55-1.70 (m, 1H) 1.77 (d,J=9.54 Hz, 3H) 1.84-1.90 (m, 1H) 2.03 (d, J=8.07 Hz, 1H) 2.31-2.41 (m,1H) 2.79-2.87 (m, 1H) 2.89-2.96 (m, 1H) 3.08 (s, 2H) 3.12-3.18 (m, 3H)3.19-3.24 (m, 1H) 3.34 (d, J=4.77 Hz, 2H) 3.41-3.45 (m, 2H) 3.46-3.55(m, 1H) 3.65 (d, J=11.37 Hz, 1H) 3.73 (dd, J=15.41, 8.44 Hz, 4H)4.26-4.40 (m, 1H) 4.48 (d, J=7.70 Hz, 1H) 4.52-4.62 (m, 1H) 4.69 (br.s.,1H) 4.81 (d, J=7.70 Hz, 1H) 4.88 (br.s., 1H) 4.91-5.03 (m, 1H) 5.05-5.26(m, 2H) 5.51 (d, J=7.70 Hz, 1H) 5.55 (br.s., 1H; the formula isC32H50O13; formula weight is 642.7316).

The NMR data obtained for the tri-glycosylated steviol glycoside are asfollows:

1H NMR (600 MHz, DMSO-d₆) δ ppm 0.73-0.79 (m, 1H) 0.81 (s, 3H) 0.89-0.97(m, 2H) 0.99-1.05 (m, 1H) 1.17 (s, 3H) 1.19 (d, J=11.37 Hz, 1H) 1.24 (s,2H) 1.31-1.40 (m, 4H) 1.40-1.51 (m, 3H) 1.55-1.63 (m, 1H) 1.67 (dd,J=14.12, 5.32 Hz, 1H) 1.71-1.82 (m, 5H) 1.88 (d, J=11.00 Hz, 1H)1.98-2.08 (m, 2H) 2.23-2.30 (m, 1H) 2.94 (t, J=8.44 Hz, 1H) 3.01-3.11(m, 3H) 3.12-3.17 (m, 1H) 3.19-3.28 (m, 3H) 3.44-3.52 (m, 5H) 3.54-3.60(m, 1H) 3.61-3.71 (m, 3H) 4.36 (br. s., 1H) 4.49 (br. s., 1H) 4.55 (d,J=7.70 Hz, 1H) 4.69 (s, 1H) 4.73 (br. s., 1H) 4.88 (br. s., 1H)4.91-5.05 (m, 2H) 5.17 (br.s., 1H) 5.31 (br.s., 1H) 5.44 (d, J=8.07 Hz,1H) 5.55 (br. s., 1H; the formula is C38H60O18; formula weight is804.8722).

The di-glycosylated steviol glycoside ester was determined to be ananalog of steviol-1,2-bioside (FIG. 8B), and the tri-glycosylatedsteviol glycoside was determined to be an isomer of RebB, both of whichare glycosylated at the 19-O position (FIG. 8C) instead of the 13-Oposition of their respective isomers. This data suggests that thesecompounds form when the activity of UGT85C is low compared to theactivity of EUGT11, UGT76G1, or UGT74G1.

Example 6: Engineering and Fermentation of EFSC 3261

The wild type Saccharomyces strain utilized in Example 1 was modified tocontain the heterologous genes in Table 14 involved in steviol glycosideproduction. The genes were all integrated into the chromosome of thehost strain using similar methods described in Example 1.

TABLE 14 List of Recombinant Genes and Promoters Used in Strain EFSC3261. Number Heterologous pathway gene of copies Promoter(s) used GGPPS7(Synechococcus sp) 2 TEF2, GPD1 synthetic gene CDPS (truncated, Zeamays) 3 PGK1 (X2), TPI1 native gene KS5 (A. thaliana) native gene 3TPI1, PDC1 (X2) KO (S. rebaudiana KO1) 2 FBA1, GPD1 synthetic gene ATR2synthetic gene 1 PGK1 KAH (S. rebaudiana KAHe1) 3 GPD1, TEF1 (X2)synthetic gene S. rebaudiana CPR 8 native 2 TPI1 (X2) gene 85C2 (S.rebaudiana) 1 GPD1 synthetic 74G1 native (S. rebaudiana) 1 TPI1 76G1synthetic (S. rebaudiana) 1 TEF1 91d2e-b 2X mutant from 1 GPD1 S.rebaudiana EUGT11 synthetic 1 TEF2 (Oryza sativa)

Fed-batch fermentation was carried out aerobically in 2 L (workingvolume) fermenters which included a ˜16 hour growth phase in the basemedium (minimal medium containing glucose, ammonium sulfate, tracemetals, vitamins, salts, and buffer) followed by ˜100 hours of feedingwith a glucose-containing defined feed medium. Glucose was utilized asthe carbon and energy source and combined with trace metals, vitamins,and salts. The pH was kept near pH 5 and the temperature setpoint was30° C. The feed rate was controlled to prevent oxygen depletion and tominimize ethanol formation (glucose-limited conditions). Whole culturesamples (without cell removal) were taken and boiled in an equal volumeof DMSO for total glycosides levels.

FIG. 9 shows production of RebD by EFSC 3261 in four separate trials.Total production (intracellular and extracellular combined) averagedtiter were between 800-1200 mg/L. For fermentation run 58, the finaltotal titer at 123 hours was 1109 mg/L RebD, 695 mg/L RebM; the ratio ofD:M on a mass basis was 1.6. 394 mg/L RebA were also produced.

Example 7: Strain Engineering and Fermentation of EFSC 3297 forIncreased Production of RebD and RebM

The same wild type Saccharomyces strain utilized in Example 1 wasmodified to contain the heterologous genes in Table 15 involved insteviol glycoside production. The genes were all integrated into thechromosome of the host strain using similar methods described inExample 1. Although the genes used are identical to those in Example 1,increased copy numbers of bottleneck enzymes in the steviol pathwayallowed for increased production of RebD and RebM. Fermentation ofstrain 3297 was carried out in a manner similar to that described abovefor strain 3261.

TABLE 15 List of Recombinant Genes and Promoters Used in Strain EFSC3297. Number Heterologous pathway gene of copies Promoter(s) used GGPPS7(Synechococcus sp) 3 TEF2 (X2), GPD1 synthetic gene CDPS (truncated, Zeamays) 4 PGK1 (X3), TPI1 native gene KS5 (A. thaliana) native gene 4TPI1, PDC1 (X3) KO (S. rebaudiana KO1) 2 FBA1, GPD1 synthetic gene ATR2synthetic gene 1 PGK1 KAH (S. rebaudiana KAHe1) 4 GPD1 (X2), TEF1 (X2)synthetic gene S. rebaudiana CPR 8 native 3 TPI1 (X3) gene 85C2 (S.rebaudiana) 1 GPD1 synthetic 74G1 native (S. rebaudiana) 1 TPI1 76G1synthetic (S. rebaudiana) 1 TEF1 91d2e-b 2X mutant from 1 GPD1 S.rebaudiana EUGT11 synthetic 2 TEF2 (X2) (Oryza sativa)

Production of RebD and RebM by EFSC 3297 is shown in FIG. 10. The ratioof D:M on a mass basis was 1.1. 1517 mg/L RebD was produced at the endof the fermentation (total, intracellular plus extracellular) and 1375mg/L of RebM was produced.

Example 8: Strain Engineering and Fermentation of EFSC 3841 with TwoCopies of the UGT76G1 Gene

The wild type Saccharomyces strain utilized in Example 1 was modified tocontain the heterologous genes in Table 16 involved in steviol glycosideproduction. The genes were all integrated into the chromosome of thehost strain using similar methods described in Example 1. Fermentationconditions for 3841 were similar to those described above for strain3261.

TABLE 16 List of Recombinant Genes and Promoters Used in Strain EFSC3841. Number Heterologous pathway gene of copies Promoter(s) used GGPPS7(Synechococcus sp) 3 TEF2 (X3) synthetic gene CDPS (truncated, Zea mays)4 PGK1 (X4) native gene KS5 (A. thaliana) native gene 4 PDC1 (X4) KO (S.rebaudiana KO1) 3 FBA1, GPD1, TPI1 synthetic gene ATR2 synthetic gene 2PGK1 (X2) KAH (S. rebaudiana KAHe1) 4 GPD1, TEF1 (X3) synthetic gene S.rebaudiana CPR 8 native 3 TPI1 (X3) gene 85C2 (S. rebaudiana) 2 GPD1(X2) synthetic 74G1 native (S. rebaudiana) 2 TPI1 (X2) 76G1 synthetic(S. rebaudiana) 2 TEF1 (X2) 91d2e-b 2X mutant from 2 GPD1 (X2) S.rebaudiana EUGT11 synthetic 2 TEF2, TEF1 (Oryza sativa)

Production of RebD, RebM, and RebA by EFSC 3841 is shown in FIG. 11.Here, the total amount of RebD produced was 2786 mg/L, and the totalamount of RebM produced was 2673 mg/L for a ratio of 1.04 D:M (g per g).703.7 mg/L RebA was also produced.

Example 9: Knockdown of One UGT76G1 Gene from EFSC 3841 and DecreasedProduction of RebD and RebM

An auxotrophic (leu2, ura3) version of strain EFSC 3841 described above,designated EFSC 3643, was further modified to delete one of the wildtype 76G1 UGT genes. The performance of 3 colonies containing one copyof UGT 76G1 was tested versus 4 colonies of the unmodified strain whichcontains 2 copies of UGT 76G1. PCR was used to verify that the newstrain only harbored one copy of the 76G1. Briefly, the disruption ofone copy of UGT76G1 was verified by two PCR reactions amplifying aregion upstream of the insertion site with part of the integrationcassette and a region downstream of the insertion site with part of theintegration cassette used for disruption. PCR primers designed for thewildtype 76G1 confirmed that wildtype 76G1 was still intact and presentin the strain. The colonies were grown in 96 deep-well plates for 96hours at 30° C. and 400 RPM. The total amounts of RebD and RebM weredetermined by LC/MS analysis.

From FIG. 12, it can be seen that the copy number of 76G1 significantlychanges the RebD/RebM ratio. The ratio of RebD to RebM was plotted forthe 3 colonies containing only one 76G1 copy (bars on the left-hand sideof the graph), versus 4 colonies of the parent strain that contained 2copies of the 76G1 UGT (right-hand bars of the graph).

Example 10: Determination of Relative Rates of RebD and RebM Production

p416GPD containing VVT-76G1 was expressed in the protease deficientyeast strain DSY6 for 48 h in SC-ura media. 100 μL of cells were thenreinoculated in 3 mL of SC-ura media for 16 h. The cells were lysed with200 μL CelLytic™ Y according to manufacturer's description. 6 μL of thelysate were added to 24 μL of the reaction mixture consisting of 20 mMTris-buffer (pH 8.0), 0.3 μMUDPG, and 0.1 μM Reb D or Reb E. Thereactions were incubated at 300C and stopped at 0, 1, 2, and 18 h bytransferring 25 μL of the 30 μL reaction mixture to 25 μL DMSO. Amountsof RebD, RebE, and RebM were analyzed by LC-MS and assessed by peakintegration during data processing as “area under the curve.”

FIG. 13 shows that a large portion of RebD is consumed withoutgenerating a corresponding amount of RebM. It is also shown that RebE isconsumed within 2 h and converted to RebD. This finding confirms analternative glycosylation route from steviol to RebM through RebEinstead of RebA is possible, which is first observed at the 18 h timepoint.

Example 11: Prediction of Amino Acids Involved in RebM and RebD Bindingin UGT76G1

As a means for identifying UGT76G1 variants with increased activity andregioselectivity towards RebM or RebD, homology modeling and dockingstudies were performed. Three homology models were generated usingstandard setting in the SybylX program with a combination of thefollowing PDB-files 2PQ6 (% ID=31), 2C1×(% ID=28), 3HBF (% ID=28), 2VCE(% ID=35) as templates. The ligands present in PDB2VCE were used duringthe generation of main- and side-chains but removed prior to energyminimization. To yield the highest quality structures, models wereenergy minimized using an AMBER FF99 forcefield with either the standardsettings or a gradient termination with a threshold of 0.1 kJ, a cutoffradius of 10 Å, and a maximum iteration of 5000 cycles. Statistics forthe models are shown in Table 17, and variance between the models can befound in FIG. 14.

TABLE 17 Summary of UGT76G1 homology models. Statistic Model 1 Model 2Model 3 Goal Clashscore,  1.1   0.42   3.38 all atoms (99^(th) (99^(th)(97^(th) percentile) percentile) percentile) Poor rotamers 16  6 8  <1%(4.47%) (1.52%) (2.02%) Ramachandran 5 12  6 <0.05%  outliers (1.26%)(2.70%) (1.35%) Ramachandran 354  375  404  >98% favored (88.94%) (84.27%)  (90.79%)  MolProbity score   1.89   1.46   1.89 (81^(st)(96^(th) (81^(st) percentile) percentile) percentile)

viations >0.25 3 1 5 0 Å (0.80%) (0.24%) (1.20%) Bad backbone bonds0/1599 0/1780 0/1780  0%   (0%)   (0%)   (0%) Bad backbone 0/1996 1/22240/2224 <0.1%  angles   (0%) (0.04%)   (0%)

indicates data missing or illegible when filed

After model generation, substrates were docked into the active site ofthe enzyme using the Surflex Dock suite in SybylX to predict the aminoacids forming the binding pocket. The UDPG portion of the UGT76G1binding groove was located by aligning the 76G1 models with PDB2VCE andimporting the ligand, UDPF2G, directly from the template. To dock theacceptor substrates, a protocol was generated using standard valuescovering the remaining part of the binding site. The dockings wereperformed using the GeomX settings on a ligand library containingsteviol glycosides allowed with protein flexibility (model 1) or noflexibility (model 2). The docking results were analyzed using acombination of the scoring functions in SybylX using top 3 dockingresults in base mode and top 1 docking result with protein flexibility.

All UGT76G1 amino acids are shown in Table 18 below. The sites for thesaturation library were determined by selecting all residues found to bewithin 5 Å of RebD and RebM in the docking analysis on two or moremodels (shown as bold “x”). Furthermore, all residues found to be within5 Å of the RebM and RebD 19-O-glucose moiety, which were positioned inthe binding site for the RebD

RebM reaction, were selected. Residues completely conserved betweensimilar enzymes, which are shown in bold and with a “!,” were omittedfrom the screen.

TABLE 18 Prediction of amino acids involved in RebM and RebD binding inUGT76G1. Enzyme UGT76G1 Top3 Top3 Top1 Top1 Top1 Model (base) (Base)(PF) (PF) (Base) 1 + 2 Unique Model no. 2 1 1 1 1 minus 5Å: Unique TotalRebM RebD cons. RebM RebD screened Substrate: RebM RebM RebM 19Glcs19Glcs AAs: 19G1c: 19Glcs: residues: Residue A: 5 5 5 5 5 5 5 5 5 No. ofres 73 58 42 15 11 23 12 3 38 in group: VAL 20 20 20 20 X x PRO 21! 2121 21 21 21 PHE 22 22 22 22 22 22 X x GLN 23 23 23 23 23 x x GLY 24 2424 24 24 X x HIS 25! 25 25 25 ILE 26 26 26 26 x x ASN 27 27 27 THR 48 48ASN 49 49 49 49 49 x x PHE 50 50 50 50 50 x x ASN 51 51 51 51 51 x x PRO53 53 53 53 x x LYS 54 54 54 54 54 x x THR 55 55 55 55 55 x x SER 56 5656 56 56 x x PRO 80 80 THR 81 81 HIS 82 82 GLY 83 83 PRO 84 84 LEU 85 8585 X x MET 88 88 ARG 89 89 ILE 92 92 GLU 95 95 95 HIS 96 96 96 ASP 99 9999 ARG 103 103 103 THR 123 123 ASP 124! 124 124 ALA 125 125 LEU 126 126126 126 X x TRP 127 127 127 127 X x TYR 128 128 128 X x VAL 143 143 LEU144 144 MET 145 145 145 X x THR 146 146 146 146 X x SER 147 147 147 147X x SER 148 148 PHE 150 150 ASN 151 151 151 151 X x PHE 152 152 ALA 154154 HIS 155 155 155 155 X x VAL 156 156 SER 157 157 LEU 158 158 PRO 159159 GLN 160 160 PHE 161 161 ASP 162 162 GLU 163 163 GLY 165 165 TYR 166166 LEU 167 167 ASP 168 168 ASP 189 189 ILE 190 190 LYS 191 191 191 191X x SER 192 192 ALA 193 193 TYR 194 194 SER 195 195 195 195 X x ASN 196196 TRP 197 197 GLN 198 198 198 198 X x ILE 199 199 199 X x LEU 200 200200 200 X x LYS 201 201 GLU 202 202 ILE 203 203 203 203 X x LEU 204 204204 204 X x GLY 205 205 LYS 206 206 MET 207 207 207 ILE 208 208 LYS 209209 SER 253 253 253 253 253 x x LEU 257 257 257 257 x x PHE 281! 281 GLY282! 282 282 282 SER 283 283 283 283 283 x x THR 284 284 284 284 284 X xSER 285 285 285 285 x x GLU 286 286 VAL 309 309 ARG 311! 311 PHE 314 314314 314 314 x x LYS 337 337 337 x x TRP 338! 338 338 338 338 HIS 356!356 GLY 358! 358 TRP 359! 359 359 ASN 360! 360 PHE 377 377 GLY 378 378378 X x LEU 379 379 379 379 X x ASP 380 380 380 380 X x GLN 381! 381 381381 PRO 382 382 LEU 383 383 ASN 384 384 Bold indicates completeconservation in amino acid. Bold “!” indicates amino acid residues thatare completely conserved between similar enzymes and were omitted fromthe screen.

Example 12: UGT76G1 Site Saturation Library Prescreen

Prior to performing the UGT76G1 site saturation library screening asdescribed herein, culture growth and production of RebM and RebD weremonitored in 96 and 4×24 deep-well plates. Using the standard lithiumacetate protocol, the EFSC 3385 strain was transformed with p416GPDcontaining VVT-76G1, and the transformants were plated on SC-URA plates.EFSC 3385 is a strain that is deficient in UGT76G1 and thus will makeRebE until transformed with a plasmid containing an active UGT76G1. Thestrain contained a disruption in the UGT76G1 coding region, which wasreplaced with the spHIS5 marker, and also contained integrated copies ofthe UGT91D2e-2× mutant, UGT74G1, ATR2, UGT85C2, S. rebaudiana CPR8 (2copies), A. thaliana KS5 (2 copies), Synechococcus GGPPS7, codonoptimized S. rebaudiana KAHe1 (2 copies), S. rebaudiana KO (two copies),the truncated Zea mays CDPS5 (2 copies), and EUGT11.

For the 96 deep-well plate condition, 96 colonies were transferred to aplate containing 1 mL SC-URA, and the plate was incubated at 300C and400 RPM for 96 h. For the 4×24 deep-well plate condition, 96 colonieswere transferred to a plate containing 3 mL SC-URA. The plate wasincubated at 300C and 320 RPM for 96 h, and 200 μL were then transferredfrom each well to a 96 deep-well plate.

50 μL of the cultures from each plate were transferred to 96 wellpolymerase chain reaction (PCR) plates and diluted 1:1 with 100%dimethyl sulfoxide (DMSO). The plates were heat sealed, incubated at800C for 10 min, and subsequently cooled to 25° C. The plates were spunat 4000 RPM for 10 min, and 50 μL of the culture mixtures weretransferred to a new plate for LC-MS analysis.

Results of the UGT76G1 site saturation library prescreen can be found inFIG. 15. Variance in RebD and RebM production can be explained byevaporation, particularly in the wells located at the edges of theplate, over the course of the 96 h incubation period. The higherconcentrations of RebM and RebD produced by colonies grown in 96deep-well plates suggest that these plates are better suited for LC-MSanalysis, as compared to 4×24 deep-well plate, and were thus selectedfor use in the UGT76G1 site saturation library screen.

Example 13: UGT76G1 Site Saturation Library Screen

Through the company, Baseclear, UGT76G1 was subcloned from EPSC2060(p423GPD) to EPSB492 (p416GPD) using the SpeI and XhoI restrictionsites, and the site saturation libraries were created using degenerateNNS-primers. Using the standard lithium acetate protocol, the EFSC3385strain was transformed with the library or with control plasmidcontaining VVT-76G1, and the transformants were plated on SC-URA plates.

1 mL of SC-URA media was added to 96 deep-well plates, and colonies fromeach of the 38 site saturation library residues identified in Example 9were picked and incubated in the 96 deep-well plates at 300C and 400 RPMfor 96 h. 50 μL of each culture samples were then transferred to 96 wellPCR plates containing 50 μL 100% DMSO. The plates were then heat sealed,incubated at 800C for 10 min, subsequently cooled to 120C, and spun at4000 RPM for 10 min. 70 μL of each supernatant were transferred to a newplate for LC-MS analysis.

FIG. 16 shows all data points of the UGT76G1 site saturation libraryscreen, with wild type production depicted with black triangles. Thevariant numbering system can be found in Table 19.

TABLE 19 Numbering for UGT76G1 site saturation library variants. NumberResidue 1 VAL 20 2 PHE 22 3 GLN 23 4 GLY 24 5 ILE 26 6 ASN 49 7 PHE 50 8ASN 51 9 PRO 53 10 LYS 54 11 THR 55 12 SER 56 13 LEU 85 14 LEU 126 15TRP 127 16 TYR 128 17 MET 145 18 THR 146 19 SER 147 20 ASN 151 21 HIS155 22 LYS 191 23 SER 195 24 GLN 198 25 ILE 199 26 LEU 200 27 GLU 202 28ILE 203 29 SER 253 30 LEU 257 31 SER 283 32 THR 284 33 SER 285 34 PHE314 35 LYS 337 36 GLY 378 37 LEU 379 38 ASP 380

Table 20 and FIG. 17 show the UGT76G1 variant colonies with the highestselectivity towards production of either RebM or RebD, which wereselected for further study. In FIG. 17, all data points with the “VVT”prefix indicate RebM and RebD production of the wild type enzyme. It isshown that selected enzyme variants exhibited an inhibited RebD to RebMactivity or an increased production of RebM, as compared to wild typecontrols.

TABLE 20 Top RebM- and RebD-producing colonies. RebM RebD Colony (μM)(μM) A1 16.28 23.35 A2 4.83 24.12 A3 2.92 25.13 A4 34.07 12.24 A5 5.6623.72 A6 11.08 23.50 A7 5.33 24.35 A8 38.36 11.90 A9 42.18 13.76 A1033.80 8.95 A11 34.66 10.13 A12 40.44 9.30 B1 36.86 13.64 B2 34.88 9.05B3 38.61 6.64 B4 20.84 24.42 B5 22.70 23.28 B6 35.49 9.63 B7 34.55 8.71B8 35.47 11.31 B9 35.62 9.17 B10 37.59 7.80 B11 36.76 12.41 B12 2.3126.75 C1 2.21 23.66 C2 9.24 24.56 C3 1.93 23.18 C4 10.47 24.70 C5 4.9123.09 C6 2.38 27.11 C7 11.06 28.32 C8 13.77 23.07 C9 35.58 9.92 C1033.51 5.02 C11 33.87 4.24 C12 6.04 30.20 D1 33.51 4.93 D2 43.18 12.50 D334.04 8.06 D4 35.92 12.38 D5 37.11 7.14 D6 44.66 11.19 D7 33.61 13.36 D836.19 8.68 D9 36.88 16.12 D10 11.25 30.00 D11 11.68 31.55 E1 56.60 7.58E2 12.18 30.41 E3 13.75 33.16 E4 8.79 27.90 E5 8.69 25.33 E6 11.78 28.56E7 8.31 23.67 E8 9.19 25.35 E9 7.77 27.41 E10 8.96 24.06 E11 11.25 31.88E12 10.18 24.20 F1 9.94 23.65 F2 38.36 12.57 F3 37.37 15.35 F4 8.8925.59 F5 10.78 23.51 F6 11.41 27.57 F7 10.96 26.18 F8 35.86 14.67 F95.69 29.07 F10 13.84 32.85 F11 2.81 27.27 F12 39.86 10.64 G1 37.94 8.80G2 9.56 23.64 G3 33.80 9.62 G4 3.01 25.15 G5 9.81 25.41 G6 42.71 9.96 G71.65 24.20 G8 9.63 24.28 G9 33.65 22.69 G10 34.87 9.69 G11 33.66 11.49G12 3.58 24.04 H1 33.94 9.84 H2 10.39 23.59 H3 43.64 9.40 H4 33.50 8.73H5 36.32 9.39 H6 34.61 7.69 H7 36.73 9.20 H8 35.49 4.09 H9 34.25 4.82H10 35.36 4.10 H11 19.87 25.19

Example 14: UGT76G1 Site Saturation Library Rescreen and VariantSequencing

A rescreen of the 47 UGT76G1 variant colonies producing either thehighest amounts of RebD or RebM was done in triplicate and showed thesame trends as the initial screen (FIG. 19). The colonies to besequenced were selected by compiling the results from the screen andrescreen. As the production levels of the screen and rescreen could notbe directly compared, the colonies were ranked from highest producers tolowest producers of RebD and RebM, respectively, and the ranks from thescreen and rescreen were averaged. From the averages, the top 16 RebD-,top 16 RebM-, and top 16 RebD/M-producing colonies (a total of 48colonies) were identified. As some of the top RebD and top RebD/Mproducers were found to be the same colonies, duplicate colonies werecounted only once, and additional colonies were chosen to reach the 48total colonies to be sequenced. These colonies were then sequenced induplicate with the GPDseq_fwd and CYC1seq-rev primers, shown below.

GPDseq_fwd primer seq: (SEQ ID NO: 88) CGG TAG GTA TTG ATT GTA ATTCYC1seq_rev primer seq: (SEQ ID NO: 89) CTT TTC GGT TAG AGC GGA TGT

Tables 21-23 show the amounts and rankings of RebD, RebM, and RebD/RebMproduced by the indicated variants, and Table 24 summarizes themutations that selectively increase either RebD or RebM production.Amounts of RebM, RebD, RebA, Rubusoside, and RebB produced by wild typeand UGT76G1 variant colonies are shown in Table 25.

TABLE 21 Identities of top RebD-producing UGT76G1 variants. RescreenScreen Sample 1 Sample 2 Sample 3 Average RebD RebD RebD RebD RebDColony (μM) Rank (μM) Rank (μM) Rank (μM) Rank (μM) RANK Mutation E225.33 21 17.00 21 18.99 7 19.35 3 20.17 13 L257A E5 33.16 1 17.51 1515.34 29 18.49 8 21.13 13 L257G F9 29.07 8 21.05 3 17.23 15 13.89 2820.31 14 L257T G5 25.15 23 21.53 2 19.07 4 14.50 25 20.06 14 S283G E328.56 9 16.42 22 — — 18.28 10 21.09 14 L257W C7 27.11 15 18.64 8 14.6432 18.97 4 19.84 15 T146A F6 25.59 18 18.17 11 15.55 25 18.87 5 19.55 15L257R, (S389F) C5 28.32 10 — — — — 16.03 20 22.18 15 T146A D11 30.00 7 —— 15.58 24 — — 22.79 16 L257R A5 23.72 35 18.52 10 16.76 19 18.66 619.42 18 I26F F1 24.06 33 19.91 4 16.90 17 — — 20.29 18 L257G B12 24.5626 18.11 12 17.29 14 15.98 21 18.99 18 T146G E4 30.41 5 16.30 23 17.4611 12.88 34 19.26 18 L257P E10 23.65 38 — — 19.31 3 17.12 15 20.03 19L257G F4 12.57 55 19.36 5 17.43 13 18.55 7 16.98 20 L257E E9 24.20 3117.49 16 16.93 16 — — 19.54 21 L257G E7 23.67 36 19.24 6 15.51 26 16.7417 18.79 21 L257G H2 23.59 40 15.85 26 17.95 8 17.63 12 18.75 22 S285RG7 24.20 30 — — 16.34 21 17.22 14 19.26 22 S283N C12 30.20 6 — — 14.5033 14.36 27 19.68 22 H155R C1 26.75 16 15.32 27 16.30 22 14.43 26 18.2023 T146G A6 23.50 42 14.96 29 19.59 2 16.25 19 18.57 23 I26W C4 24.70 25— — — — 15.94 22 20.32 24 T146P

TABLE 22 Identities of the top RebM-producing UGT76G1 variants. RescreenScreen Sample 1 Sample 2 Sample 3 Average RebD RebD RebD RebD RebDColony (μM) Rank (μM) Rank (μM) Rank (μM) Rank (μM) RANK Mutation G137.94 12 — — 26.68 7 26.77 6 30.46 8 T284G H7 36.73 19 — — 29.81 1 25.1112 30.55 11 K337P B1 36.86 17 — — — — 27.82 5 32.34 11 T55K D2 43.18 421.39 30 27.28 4 — — 30.62 13 Q198R H3 43.64 3 23.32 21 24.67 14 22.0323 28.42 15 S285T C11 33.87 39 28.92 3 26.07 8 24.21 14 28.27 16 H155LA10 33.80 41 25.52 10 23.48 15 29.50 1 28.08 17 S56A B11 36.76 18 25.2812 20.88 28 24.69 13 26.90 18 Y1285 H04 33.50 47 25.34 11 24.98 13 28.713 28.13 19 K337E B02 34.88 30 28.40 4 — — 20.65 27 27.98 20 T55E D0956.60 1 21.14 32 20.84 29 — — 32.86 21 S253G D01 33.51 45 23.48 20 25.2711 25.33 10 26.89 22 H155L H09 34.25 35 — — 21.90 21 25.28 11 27.14 22L379V G11 33.65 43 22.95 22 27.77 3 — — 28.12 23 T284R B8 35.47 28 25.669 21.01 26 20.13 28 25.57 23 Y128E F2 11.41 58 31.01 1 21.06 25 26.62 722.53 23 S253W C10 33.51 46 22.04 28 25.54 10 25.42 8 26.63 23 H155L

TABLE 23 Identities of the top RebD/M-producing UGT76G1 variants. Aver-Aver- Aver- Aver- Rank age age age age RebD + RebD Rank RebM Rank RankColony (μM) RebD (μM) RebM (96-M) Mutation G7 19.26 22 1.51 84 34 S283NF9 20.31 14 5.27 71 38 L257T C1 18.20 23 1.90 79 40 T146G B12 18.99 183.74 74 41 T146G A5 19.42 18 6.76 68 46 I26F F6 19.55 15 8.23 63 48L257R + S389F C6 17.74 29 2.59 77 48 T146G F1 20.29 18 7.98 65 49 L257GE2 20.17 13 8.64 60 49 L257A C7 19.84 15 7.52 61 50 T146A C3 16.30 371.74 83 50 T146G E11 17.80 25 6.48 69 52 L257G A2 15.34 32 4.48 76 52Q23H E5 21.13 13 9.32 57 52 L257G E4 19.26 18 8.00 61 54 L257P G5 20.0614 8.79 56 54 S283G D11 22.79 16 9.75 58 54 L257R C5 22.18 15 10.81 5754 T146A E9 19.54 21 8.21 62 55 L257G E7 18.79 21 8.00 62 55 L257G C218.29 26 6.95 67 55 T146A A3 15.25 35 3.19 76 56 Q23G E12 17.28 25 7.2665 56 L257W E10 20.03 19 10.10 57 57 L257G

TABLE 24 Summary of UGT76G1 variants for RebD production and RebMproduction. RebD Q23G, Q23H, I26F, I26W, T146A, T146G, T146P, H155R,L257P, L257W, L257T, L257G, L257A, L257R, L257E, S283G and S283N RebMT55K, T55E, S56A, Y128S, Y128E, H155L, H155R, Q198R, S285R, S285T,S253W, S253G, T284R, T284G, S285G, K337E, K337P and L379V

TABLE 25 Production of steviol glycosides by UGT76G1 variants. RebM RebDRebA Rubu RebB Total RebA-->RebM Muta- AVG SUM tion (μM) WT 22.35 4.987.03 0.65 2.54 34.37 WT 24.01 5.14 5.68 0.77 2.43 34.83 RebD-optimizingmutations L257W 9.35 18.46 4.70 2.03 32.52 L257A 8.62 18.45 4.75 1.9731.82 L257E 8.72 18.44 3.70 1.54 30.87 L257G 7.49 18.40 4.44 0.18 1.8030.34 S283G 10.71 18.37 3.77 1.58 32.85 L257G 10.18 18.22 4.47 0.20 1.8732.86 I26F 7.13 17.98 3.81 1.64 28.92 Q23H 4.30 17.88 3.04 1.38 25.22L257R, 8.01 17.53 4.21 1.92 29.75 S389F T146A 9.24 17.41 4.16 0.18 1.7230.82 L257T 5.13 17.39 2.73 0.25 1.29 25.24 L257W 11.40 17.35 4.28 1.9433.04 L257G 7.23 17.21 4.19 0.45 1.81 28.62 L257G 7.90 17.16 4.16 1.7329.22 S285R 8.66 17.14 3.44 1.70 29.25 T146G 1.91 17.13 1.69 0.74 20.73L257G 7.84 17.12 3.93 1.80 28.88 I26W 11.51 16.93 5.22 1.93 33.67 S283N1.45 16.78 1.48 0.67 19.70 T146A 10.57 16.03 4.07 1.86 30.68 T146G 1.8215.95 1.75 0.22 0.72 19.52 T146P 10.12 15.94 4.05 1.96 30.11 T146A 9.3115.60 4.34 1.91 29.25 L257R 8.26 15.58 3.60 0.17 1.88 27.44 L257P 6.6115.55 3.62 0.15 1.59 25.78 T146G 1.76 15.35 1.81 0.24 0.68 18.92 H155R10.49 14.43 4.46 0.25 1.78 29.38 L257G 5.12 14.03 3.63 0.95 22.78 T146G1.64 12.86 1.65 0.21 0.67 16.14 Q23G 3.33 11.96 1.71 0.16 0.80 17.00RebM-optimizing mutations T55K 27.82 7.96 6.28 0.40 2.31 42.06 K337P27.46 8.14 5.83 0.41 2.18 41.43 T284G 26.72 7.29 7.27 0.34 2.72 41.28H155L 26.40 3.70 6.79 0.30 2.50 36.89 K337E 26.34 7.14 6.35 0.39 2.6639.83 S253W 26.23 6.83 5.82 0.37 2.26 38.89 S56A 26.17 6.50 6.94 0.662.46 39.61 T284R 25.36 7.99 6.24 0.24 2.56 39.58 H155L 24.69 3.65 7.380.39 2.66 35.72 T55E 24.52 6.73 5.84 0.46 2.09 37.09 Q198R 24.33 6.616.37 0.60 2.33 37.32 H155L 24.33 4.00 7.43 0.29 2.53 35.76 Y128S 23.626.20 6.48 0.43 2.29 36.29 L379V 23.59 4.01 7.09 0.28 2.72 34.69 S285T23.34 5.90 5.73 0.44 2.30 34.97 Y128E 22.27 5.04 5.78 0.41 2.17 33.09S253G 20.99 7.26 5.78 0.27 2.42 34.04

Example 15: Determination of Relative Rates of UGT76G1 GlycosylationReactions

UGT76G1 was only known in the literature to catalyze the1,3-glycosylation of 1,2-stevioside to convert it into RebA andconverting 1,2-bioside to RebB. The inventors have newly discovered thatthe reactions shown in Table 26 are catalyzed by UGT76G1.

TABLE 26 Newly discovered UGT76G1 reactions. Substrate ProductRebaudioside D Rebaudioside M Rubusoside “Rebaudioside Q”(1,3-O-glycoside linkages on both the 13- and 19-O-glucose position)Steviol 1,2 bioside Rebaudioside B isomer (19-O) isomer (19-O)

Similar to Example 9, p416GPD containing VVT-76G1 was expressed in theprotease deficient yeast strain DSY6 for 48 h in SC-ura media. 100 μL ofcells were then reinoculated in 3 mL of SC-ura media for 16 h. The cellswere lysed with 200 μL CelLytic™ Y according to manufacturer'sdescription. 6 μL of the lysate were added to 24 μL of the reactionmixture consisting of 20 mM Tris-buffer (pH 8.0), 0.3 μMUDPG, and either0.1 μM rubusoside, 0.2 μM 1,2-bioside, 0.2 μM 1,2-stevioside, 0.2 μMRebA, or 0.1 μM RebE. The reactions were incubated at 300C and stoppedat 0, 1, 2, and 18 h by transferring 25 μL of the 30 μL reaction mixtureto 25 μL DMSO. Amounts of steviol glycosides were analyzed by LC-MS andassessed by peak integration during data processing as “area under thecurve.”

In FIG. 19, it is shown that an approximately 50% decrease in the “areaunder the curve” for rubusoside resulted in considerable production of1,3-stevioside (RebG) over 18 h. RebQ, newly discovered by theinventors, was first detected at 18 h. Additionally, FIG. 19 shows that1,2-stevioside was not completely consumed over the 18 h period for theproduction of RebA as 1,2-bioside was for the production of RebB.

Furthermore, using either 1,2-stevioside or RebA as a substrate, a peakeluting at 1.96 min on the steviol+5 glucose chromatogram appeared (FIG.20). Because RebD elutes at 1.11 min and UGT76G1 only catalyzes1,3-glycosylation reactions, the peak eluting at 1.96 min appeared to beRebl. However, it was not possible to integrate the Rebl peak because itwas situated in a substrate artifact peak (FIG. 20).

These results collectively indicated that UGT76G1 preferentiallycatalyzed glycosylation of steviol glycoside substrates that are1,2-di-glycosylated on the 13-O-position, followed by steviol glycosidesubstrates that are mono-glycosylated at the 13-O-position. Thereappeared to be little preference arising from the glycosylation state ofthe 19-O-position. FIG. 1 summarizes steviol glycoside glycosylationreactions and the enzymes by which they are known to be catalyzed.

Example 16: Production of Steviol Glycosides by UGT76G1 Variants

Quantitative standards are not commercially available for each steviolglycoside, which prevented some concentration measurements. Therefore,production of additional steviol glycosides by the enzyme variants, ascompared to the wild type enzyme, was assessed by peak integrationduring data processing. The “area under the curve” data for each variantwas normalized to the wild type UGT76G1 and is shown in Table 27. Thesedata were in agreement with previous examples but also showed that someof the variants did not produce 1,3-stevioside (RebG), rubusoside, “RebQ,” and/or a steviol-tetraglycoside eluting at 1.43 min, as the wildtype controls did. Increases in RebM and RebD production can beexplained by this observation.

TABLE 27 “Area under the curve” data as a measure of the production ofsteviol glycosides by UGT76G1 variants relative to wildtype UGT76G1production. 0.95 min 1.3 at 13 Stv4Glc and 1.2 1.3 19- 13- (suspect 1.43min 19 Pos Variant Reb M Reb D RebA Stev Stev RebB Rubu 1.2 bios 1.3Bios SMG SMG Stev RebE) Stv4Glc “Reb Q” O234 0.2 2.5 0.4 2.5 — 0.5 — — —— 1.0 — 3.7 0.5 — O23G 0.2 2.5 0.3 2.0 — 0.3 0.3 — — — 0.5 — 2.8 0.4 —

0.3 3.8 0.6 2.2 — 0.7 — — — — 1.1 — 3.0 0.5 —

0.5 3.6 0.9 1.8 — 0.8 — — — — 9.6 — 2.5 0.4 —

1.2 1.4 1.2 1.2 0.7 1.0 1.8 — — — 1.2 — 1.4 0.3 0.7 T55K 1.3 1.7 1.0 1.0— 1.0 0.6 — — — 1.0 — 1.5 0.9 — T55E 1.2 1.4 1.0 1.3 0.8 0.9 0.7 — — —0.0 — 1.7 0.5 0.9 T128E 1.1 1.3 1.0 1.3 — 0.9 0.8 — — — 1.0 — 1.0 0.70.4

1.1 1.2 1.1 1.2 0.6 1.0 0.7 — — — 1.0 — 1.3 1.0 0.6 T148G 0.1 2.5 0.33.7 — 0.3 — — — — 1.0 — 7.6 0.9 — T148G 0.1 3.3 0.3 4.0 — 0.3 0.4 — — —1.4 — 7.3 1.0 — T146A 0.4 3.3 0.7 2.1 — 0.8 — — — — 0.9 — 3.2 0.6 —T146C 0.1 2.2 0.3 2.8 — 0.3 0.3 — — — 1.2 — 6.2 0.8 — T148P 0.5 3.4 0.72.2 — 0.8 — — — — 0.9 — 3.0 0.5 — I146A 0.5 3.4 0.7 2.2 — 0.8 — — — —0.2 — 3.3 0.5 — T140G 0.1 3.4 0.3 3.7 — 0.3 0.3 — — — 1.2 — 6.0 0.9 —T145A 0.4 3.7 0.2 2.2 — 0.7 0.3 — — — 1.1 — 3.8 0.5 — H155L 1.2 0.9 1.21.0 — 1.1 0.4 — — — 1.0 — 0.6 — 0.4 H155R 1.3 0.8 1.3 0.8 — 1.1 0.5 — —— 1.0 — 0.5 — 0.3 H155M 0.5 3.1 0.7 2.2 — 0.8 0.4 — — — 0.3 — 2.0 0.5 —Q198R 1.2 0.8 1.2 1.0 — 1.1 0.6 — — — 1.1 — 0.8 0.5 0.5 S253G 1.2 1.41.1 1.3 0.8 1.0 0.9 — — — 1.1 — 1.6 — 0.6 L257K 1.0 1.5 1.0 1.3 — 1.00.4 — — — 1.2 — 1.2 0.2 — L257A 0.4 3.3 0.8 2.4 — 0.8 0.3 — — — 1.1 —2.8 0.6 — L257W 0.4 3.0 0.5 2.5 — 0.8 — — — — 1.3 — 4.3 0.2 — L257P 0.63.7 0.7 2.5 — 0.8 — — — — 0.9 — 3.8 0.6 — L257G 0.3 3.3 0.0 2.3 — 0.70.2 — — — 0.9 — 3.1 0.5 — L257G 0.4 3.0 0.7 2.4 — 0.8 — — — — 1.2 — 3.30.5 — L257G 0.4 3.6 0.7 2.4 — 0.7 — — — — 1.2 — 3.6 0.6 — L257O 0.3 3.60.3 3.7 — 0.8 0.7 — — — 1.2 — 7.0 0.9 — L257G 0.5 3.9 0.3 2.8 — 0.8 0.3— — — 1.1 — 7.2 0.7 — L257G 0.2 3.0 0.6 2.2 — 0.4 — — — — 1.0 — 3.0 0.5— L257W 0.3 2.9 1.8 4.1 — 1.8 0.8 — — — 1.3 — 4.0 0.5 — L257G 0.4 3.90.7 2.6 — 0.8 0.3 — — — 1.4 — 4.1 0.7 — L257W 1.3 1.4 1.0 1.3 — 1.0 0.8— — — 0.8 — 1.4 0.7 0.4 L257E 0.4 3.9 0.6 2.4 — 0.7 — — — — 0.8 — 3.90.6 — L257R 0.4 2.7 0.7 2.5 — 0.8 — — — — 1.1 — 3.5 0.5 — S380F L257T0.2 3.7 0.5 2.6 — 0.5 0.4 — — — 0.8 — 4.0 0.6 — T287G 1.3 1.0 1.2 1.3 —1.2 0.5 — — — 1.1 — 2.2 0.7 — S283G 0.5 3.9 0.6 1.9 — 0.7 — — — — 1.1 —2.5 0.4 — S283N 0.1 3.6 0.2 3.9 — 0.3 — — — — 1.1 — 0.8 1.0 — T264R 1.21.7 1.0 1.4 — 1.1 0.4 — — — 1.0 — 2.2 0.4 — S285R 0.4 3.0 0.6 2.6 — 0.7— — — — 1.3 — 4.4 0.0 — S285T 1.1 1.3 1.0 1.0 — 1.0 0.7 — — — 1.0 — 1.20.0 0.3 K237E 1.3 1.5 1.1 1.1 — 1.1 0.6 — — — 1.2 — 1.2 0.7 — K337P 1.31.7 1.0 1.3 — 0.9 0.6 — — — 1.1 — 1.0 1.1 0.4 L329V 1.1 0.9 1.2 1.2 —1.2 0.4 — — — 1.1 — 1.1 0.4 —

indicates data missing or illegible when filed

Table 28 summarizes trends in steviol glycoside production throughRebD-optimizing and RebM-optimizing mutations, compared to thenormalized production of wild type controls. The variants with increasedRebD production appeared to primarily be the result of inhibiting theRebD→RebM reaction. Additional RebD production can stem from inhibitionof the Rubu→RebG→RebQ steviol glycosylation branch as well as areduction in RebB and of a tetraglucoside eluting at 1.43 mi. Thefour-fold increase in 1,2-Stevioside and seven-fold increase in RebEwere unexpected, but the RebE increase could be a seven-fold increase ina very small amount of sideproduct found in the wild type controls.Nevertheless, this finding indicated that the Stevioside-RebA reactionhad also been partially inhibited by the RebD-optimizing mutations,which was seen as a reduction in RebA intermediate.

TABLE 28 Steviol glycoside production by the UGT76G1 wild type or RebDand RebM-optimizing mutations. 1.3 at 13 0.95 min 1.43 min and 1.2 1.319- 13- Stv4Glc Stv4Glc 19 Pos Variant RebM RebD RebA Stev Stev RebBRubu 1.2 bios 1.3 Bios SMG SMG Stev RebE) (UNK) “Reb Q” RebD 0.1- 2.5-0.3- 1.8- 0.0x 0.3- 0.0- — — — 0.9- — 3.0-7.0x 0.4-0.9x 0.0x 0.5x 3.9x0.8x 4.0x 0.8x 0.4x 1.3x RebM 1.0- 0.8- 1.0- 0.6- 0.0- 0.8- 0.4- — — —0.9- — 0.5-2.0x 0.0-0.9x 0.0-0.8x 1.3x 1.7x 1.2x 1.3x 0.8x 1.1x 1.0x1.2x WT 23.2 5.1 6.4 AUC AUC 2.5 0.7 — — — AUC — AUC AUC AUC μM μM μM μMμM

The mutation resulting in the highest RebD levels L257G, produced nearlyfour-fold the RebD of the wild type and was found in six coloniessequenced. Other L257 mutations demonstrated nearly the sameproductivity. Mutants I26W and S283G showed the highest RebD/steviosideratio, indicating that these mutations lead to the greatest inhibitionof the RebD

M reaction without mitigating the Stev

RebA reaction. These two mutations also completely abolished the Rubu

RebG

RebQ pathway and reduced the amount of the tetraglucoside eluting at1.43 min while minimally affecting RebB production. The best RebD/RebMratios were found with T146G and S283N mutants, which showed a40-50-fold increase over the wild type. The S389Fmutation found withL257R demonstrated higher RebD production than L257R alone.

Generally, increases in RebM also resulted in increases in RebD, whiledecreasing or completely blocking the Rubu

RebG

RebQ glycosylation pathway and the tetraglucoside eluting at 1.43 min.Yet, the remaining steviol glycosides studied appeared unaffected. Thetop RebM producers, T55K and K337P, each increased RebM by 1.3 fold anddecreased rubusoside by 0.6-fold, compared to the wild type. Sincerubusoside was only present at 0.7 μM in the wild type, this observeddecrease was insufficient to explain the increase in RebM. The Rubu

RebG

RebQ pathway was almost removed, with no 1,3-stevioside (RebG) producedby these mutants. As well, the variant with the K337P mutation produced0.6-fold the levels of RebQ with the wild type. The mutations H155L andL379V each produced more RebM to RebD, with the wild type UGT76G1producing approximately 4.58 RebM per RebD, and H155L and L379 producingapproximately 6.66 and 5.88 RebM per RebD, respectively. Through thedata uncovered by this study, UGT76G1 enzymes can be screened toidentify species having improved kinetics towards RebD and RebM and thatminimize side products, thereby increasing the flux towards the desiredsteviol glycosides.

Having described the invention in detail and by reference to specificembodiments thereof, it will be apparent that modifications andvariations are possible without departing from the scope of theinvention defined in the appended claims. More specifically, althoughsome aspects of the present invention are identified herein asparticularly advantageous, it is contemplated that the present inventionis not necessarily limited to these particular aspects of the invention.

REFERENCES

-   1. Critical Reviews in Food Science and Nutrition, 52:11, 988-998,    DOI::10.1080/10408398.2010.519447.-   2. J Nat Prod. 2013 Jun. 28; 76(6):1201-28. doi: 10.1021/np400203b.    Epub 2013 May 28.-   3. Plant Physiology and Biochemistry 63 (2013) 245e253.-   4. Praveen Guleria and Sudesh Kumar Yadav, 2013. Insights into    Steviol Glycoside Biosynthesis Pathway Enzymes Through Structural    Homology Modelling. American Journal of Biochemistry and Molecular    Biology, 3: 1-19.-   5. The Plant Journal (2005) 41, 56-67 doi:    10.1111/j.1365-313X.2004.02275.-   6. Madhav et al., “Functional and structural variation of uridine    diphosphate glycosyltransferase (UGT) gene of Stevia    rebaudianaeUGTSr involved in the synthesis of rebaudioside A” Plant    Physiology and Biochemistry 63 (2013) 245e253.-   7. Jewett M C, et al., Molecular Systems Biology, Vol. 4, article    220 (2008).-   8. Masada S et al., FEBS Letters, Vol. 581, 2562-2566 (2007).

1. A method for producing Rebaudioside M (RebM) or a steviol glycosidecomposition comprising RebM, comprising contacting a startingcomposition, comprising a steviol, a steviol glycoside having a13-O-glucose, a 19-O-glucose, or both a 13-O-glucose and a 19-O-glucose,and/or a mixture thereof with a first uridine 5′-diphospho (UDP)glycosyl transferase polypeptide capable of beta 1,2 glycosylation of aC2′ of the 13-O-glucose, the 19-O-glucose, or both the 13-O-glucose andthe 19-O-glucose of the steviol glycoside and a uridine 5′-diphospho(UDP) glycosyl transferase polypeptide capable of beta 1,3 glycosylationof the C3′ of the 13-O-glucose, the 19-O-glucose, or both the13-O-glucose and the 19-O-glucose of the steviol glycoside and a numberof UDP-sugars, under suitable reaction conditions to transfer a numberof sugar moieties from the number of UDP-sugars to the steviolglycoside, thereby producing RebM or the steviol glycoside compositioncomprising RebM; wherein the first 5′-UDP glycosyl transferasepolypeptide is capable of converting Rebaudioside A (RebA) toRebaudioside D (RebD) at a rate that is at least 10 times faster thanthe rate at which a UDP glycosyl transferase polypeptide having theamino acid sequence set forth in SEQ ID NO:15 or SEQ ID NO:86 is capableof converting RebA to RebD under corresponding reaction conditions;and/or wherein the first 5′-UDP glycosyl transferase polypeptide iscapable of converting higher amounts of RebA to RebD compared to the UDPglycosyl transferase polypeptide having the amino acid sequence setforth in SEQ ID NO:15 or SEQ ID NO:86 under corresponding reactionconditions.
 2. The method of claim 1, wherein the steviol or the steviolglycoside in the starting composition is plant-derived or synthetic. 3.The method of claim 1, wherein the starting composition is furthercontacted with: (a) a second 5′-UDP glycosyl transferase polypeptidecapable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose,19-O-glucose, or both 13-O-glucose and 19-O-glucose of the steviolglycoside; and/or (b) a 5′-UDP glycosyl transferase polypeptide capableof glycosylating steviol or the steviol glycoside at its C-19 carboxylgroup; and/or (c) a 5′-UDP glycosyl transferase polypeptide capable ofglycosylating steviol or the steviol glycoside at its C-13 hydroxylgroup.
 4. The method of claim 3, wherein: (a) the second 5′-UDP glycosyltransferase polypeptide capable of beta 1,2 glycosylation of the C2′ ofthe 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose ofthe steviol glycoside comprises a polypeptide having 90% or greatersequence identity to the amino acid sequence set forth in SEQ ID NO:15or SEQ ID NO:86, a polypeptide having a substitution at residues 211 and286 of SEQ ID NO:15, or a combination thereof; and/or (b) the 5′-UDPglycosyl transferase polypeptide capable of glycosylating steviol or thesteviol glycoside at its C-19 carboxyl group comprises a polypeptidehaving 55% or greater sequence identity to the amino acid sequence setforth in SEQ ID NO:19; and/or (c) the 5′-UDP glycosyl transferasepolypeptide capable of glycosylating steviol or the steviol glycoside atits C-13 hydroxyl group comprises a polypeptide having 55% or greatersequence identity to the amino acid sequence set forth in SEQ ID NO:26;and/or (d) the 5′-UDP glycosyl transferase polypeptide capable of beta1,3 glycosylation of the C3′ of the 13-O-glucose, the 19-O-glucose, orboth the 13-O-glucose and the 19-O-glucose of the steviol glycosidecomprises a polypeptide having 50% or greater sequence identity to theamino acid sequence set forth in SEQ ID NO:2.
 5. The method of claim 1,wherein the first 5′-UDP glycosyl transferase polypeptide capable ofbeta 1,2 glycosylation of a C2′ of the 13-O-glucose, the 19-O-glucose,or both the 13-O-glucose and the 19-O-glucose of the steviol glycosidecomprises a polypeptide having 65% or greater sequence identity to theamino acid sequence set forth in SEQ ID NO:16.
 6. The method of claim 3,wherein the starting composition comprises stevioside, RebA,Rebaudioside B (RebB), Rebaudioside E (RebE), RebD, or mixtures thereof.7. The method of claim 4, wherein the 5′-UDP glycosyl transferasepolypeptide capable of beta 1,3 glycosylation of the C3′ of the13-O-glucose, the 19-O-glucose, or both the 13-O-glucose and the19-O-glucose of the steviol glycoside comprises a polypeptide having oneor more of the following amino acid substitutions of SEQ ID NO:2: Q23G,Q23H, I26F, I26W, T55K, T55E, S56A, Y128S, Y128E, T146A, T146G, T146P,H155R, H155L, H155R, Q198R, S253W, S253G, L257P, L257W, L257T, L257G,L257A, L257R, L257E, S283G, S283N, T284R, T284G, S285R, S285T, S285G,K337E, K337P, and L379V.
 8. The method of claim 1, wherein the first5′-UDP glycosyl transferase polypeptide capable of beta 1,2glycosylation of a C2′ of the 13-O-glucose, the 19-O-glucose, or boththe 13-O-glucose and the 19-O-glucose of the steviol glycoside and5′-UDP glycosyl transferase polypeptide capable of beta 1,3glycosylation of the C3′ of the 13-O-glucose, the 19-O-glucose, or boththe 13-O-glucose and the 19-O-glucose of the steviol glycoside areexpressed in a recombinant host cell.
 9. The method of claim 3, wherein:(a) the second 5′-UDP glycosyl transferase polypeptide capable of beta1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both13-O-glucose and 19-O-glucose of the steviol glycoside; and/or (b) the5′-UDP glycosyl transferase polypeptide capable of glycosylating steviolor the steviol glycoside at its C-19 carboxyl group; and/or (c) the5′-UDP glycosyl transferase polypeptide capable of glycosylating steviolor the steviol glycoside at its C-13 hydroxyl group are expressed in arecombinant host cell.
 10. The method of claim 8, wherein therecombinant host cell is capable of producing RebM when fed withrubusoside, 1,2-stevioside, or a mixture thereof.
 11. The method ofclaim 8, wherein the recombinant host cell is capable of producing RebMwhen fed with steviol-13-O-glucoside and the cell further comprises: (a)a second recombinant gene encoding the second 5′-UDP glycosyltransferase polypeptide capable of beta 1,2 glycosylation of the C2′ ofthe 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose ofthe steviol glycoside; and (b) a gene encoding the 5′-UDP glycosyltransferase polypeptide capable of glycosylating steviol or the steviolglycoside at its C-19 carboxyl group.
 12. The method of claim 8, whereinthe recombinant host cell is a fungal cell, an algal cell, or abacterial cell.
 13. The method of claim 12, wherein the fungal cellcomprises a yeast cell, and wherein the yeast cell is a cell fromSaccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowialipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii,Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candidaboidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, orCandida albicans species.
 14. The method of claim 1, which is anenzymatic in vitro method.
 15. The method of claim 8, which is a wholecell in vitro method.
 16. The method of claim 15, wherein thecomposition comprises sucrose and sucrose synthase to regenerateUDP-glucose from the UDP generated during glycosylation reactions. 17.The method of claim 15, wherein the enzymatic in vitro method comprisesmultiple reactions carried out together or stepwise.
 18. The method ofclaim 15, wherein the whole cells are in suspension or immobilized. 19.The method of claim 15, wherein the cells are permeabilized tofacilitate transfer of substrate into the cells.
 20. The claim of method19, wherein the cell are permeabilized with a solvent, a detergent, asurfactant, electroporation or slight osmotic shock.